Methods for bone treatment by modulating an arachidonic acid metabolic or signaling pathway

ABSTRACT

Methods for promoting osteogenesis to accelerate or enhance bone fracture healing, treat bone defects, and enhance bone formation are disclosed. The methods modulate an arachidonic acid metabolic or signaling pathway in general, and, in particular, utilize 5-lipoxygenase inhibitors. These molecules can be delivered alone or in combination with one or more agents that inhibit bone resorption, regulate calcium resorption from bone, enhance bone accumulation, enhance bone formation, induce bone formation, impair growth of microorganisms, reduce inflammation, and/or reduce pain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/995,529, granted as U.S. Pat. No. 7,829,535, which is a NationalStage of International Application No. PCT/US2006/032367, filed Aug. 18,2006, published in English under PCT Article 21(2), which claims thebenefit of U.S. Provisional Application Ser. No. 60/709,838, filed onAug. 18, 2005, each of which are incorporated by reference in theirentirety.

SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named “17738US_sequencelisting.txt,” created on Nov. 5,2010, with a size of 42 kb. The sequence listing consists of 43sequences and is incorporated by reference.

FIELD OF INVENTION

The invention relates generally to accelerating or enhancing boneformation or fracture healing by modulating an arachidonic acidmetabolic or signaling pathway, in particular by using inhibitors of5-lipoxygenase activity.

BACKGROUND OF THE INVENTION

Bone fractures are a common traumatic injury. Approximately 8-10 millionbone fractures are reported annually in the United States with more than1 million of these requiring hospitalization. The estimated annual costsof treating these fractures exceeds 20 billion dollars. While this isalready significant, these numbers are expected to increase due to theaging of the general population. Further, among military personnel, bonefractures are common training injuries. Bone fractures, typicallylocated in the arms and legs, are also common battle wounds. Aside fromtraumatic injury, bone fractures also can be caused by disease.Osteoporosis is caused by a reduction in bone mineral density in maturebone and results in fractures after minimal trauma. The disease iswidespread and has a tremendous economic impact. The most commonfractures occur in the vertebrae, distal radius and hip. An estimatedone-third of the female population over age 65 will have vertebralfractures, caused in part by osteoporosis. Moreover, hip fractures arelikely to occur in about one in every three woman and one in every sixmen by extreme old age.

Fracture healing is a complex tissue regeneration process that involvescell migration, proliferation, apoptosis, and differentiation inresponse to growth factors, cytokines, other signaling molecules, and tothe mechanical environment. The temporal order and magnitude of eachcellular process must be controlled for optimal regeneration. The normalevents of fracture healing are described below as occurring in 4 phases.In the initial phase, hematoma formation and localized tissue hypoxiaare the initial cellular and molecular events of fracture healing. Thesecond phase, called the early stage, is characterized by inflammationfollowed by rapid accumulation of cells at the fracture site. Thepresence of macrophages and neutrophils at the fracture site duringinflammation precedes the rapid migration and proliferation ofmesenchymal cells at the fracture site. In the third, regenerativephase, endochondral ossification creates the new bone which bridges thefracture. At this point, the fracture callus has a well-definedmorphology. Intramembraneous ossification creates buttresses ofperiosteal bone at the callus periphery. Mesenchymal cells within thecallus begin to differentiate into chondrocytes at the interface of theperiosteal bone buttress. Each new chondrocyte develops as would beexpected with matrix deposition followed by matrix calcification toproduce calcified cartilage and then apoptosis. Channels are formed intothe calcified cartilage starting at the periosteal bone buttresses.Osteoblasts migrate or differentiate on the surface of the calcifiedcartilage within these channels and begin depositing new bone. Aschondrocyte differentiation proceeds from the periphery to the center ofthe callus (fracture site), channel formation, osteoblastdifferentiation, and new bone formation follows until the soft callushas been replaced with woven (immature) bone. Angiogenesis during theregenerative phase is essential. The immature woven bone created duringthe regenerative phase is mechanically unsuited for normalweight-bearing. To compensate for the decreased mechanical properties ofthe woven bone, the fracture callus has a significantly larger diameterwhich provides for greater structural mechanical properties. In thefinal, remodeling phase, fracture callus diameter diminishes until thebone obtains its normal dimensions while maintaining the bones overallmechanical properties by enhancing material mechanical properties. Thisis accomplished by replacing the mechanically poor, woven bone withmechanically strong, lamellar (mature) bone. In successive rounds,osteoclasts resorb the woven bone and osteoblasts replace it withlamellar bone. Molecular mechanisms governing osteoclast formation andfunction occurs through the RANKL-RANK pathway and this pathway isactivated during fracture healing.

Fractures are generally treated conservatively by closed reduction ofthe fracture and immobilization (casting) of the affected bone. In suchcases, the bone heals through the endochondral ossification pathwaydescribed above. Adequate nutrition to include vitamin C, vitamin D, andcalcium aids in healing. There has been no major advancement in thetreatment of bone fractures since the mid 20^(th) century when openreduction and internal fixation of fractures became commonplace. Thepromise of growth factor treatments to enhance fracture healing has notbeen realized yet.

Unfortunately, many fractures require surgical intervention to increasehealing success and reduce the likelihood of complication. There is onlyone approved pharmacological enhancement for bone healing and that istreatment with recombinant bone morphogenetic protein, either BMP-2 orBMP-7 (OP-1). Use of these growth factors requires surgery and due toexpense and unknown potential side effects caused by the use ofsupraphysiological levels of growth factors, BMPs are used as alast-resort to heal recalcitrant fractures. Typical patient care alsoinvolves the administration of antibiotics, a narcotic, an NSAID, aCOX-2 inhibitor or other pain killers during the healing process.

NSAIDs inhibit cyclooxygenase, thereby inhibiting the conversion ofarachidonic acid into prostaglandins (PGD2, PGE2, PGF2α, PGI2, TXA2).Arachidonic acid is also a precursor for the leukotrienes (LTB4, LTC4,LTD4, LTE4), lipoxins (LXA4, LXB4), and 5-hydroxyeicosatetraenoic acid(5-HETE). The enzyme 5-lipoxygenase (5-LO) converts arachidonic acid to5-hydroperoxyeicosatetraenoic acid (5-HpETE). This is the first step inthe metabolic pathway which yields 5-HETE, the leukotrienes (LTs), andthe lipoxins. Leukotrienes are also pro-inflammatory with the ability toattract neutrophils and cause capillary permeability. The arachidonicacid metabolic pathway is summarized in FIG. 1.

Lipoxygenases are nonheme iron-containing enzymes found in plants andanimals that catalyze the oxygenation of certain polyunsaturated fattyacids, such as lipids and lipoproteins. Several lipoxygenase enzymes areknown, each having a characteristic oxidation action. Mammalianlipoxygenases are named by the position in arachidonic acid that isoxygenated. For example, the enzyme 5-lipoxygenase converts arachidonicacid to 5-hydroperoxyeicosatetraenoic acid (5-HpETE), while the enzyme12-lipoxygenas converts arachidonic acid to 12-HpETE. The activity of5-lipoxygenase requires a co-factor commonly called FLAP (fivelipoxygenase activating protein). Leukotriene synthesis is reduced bydrugs that inhibit FLAP (MK866) or mice lacking FLAP.

WO 95/30419 discloses 5-LO inhibitors reduce osteoclast activity. Thesuppression of osteoclast activity inhibits bone resorption and reducesbone loss in human pathological conditions. Bone resorption is anintegral part of fracture healing because it is necessary to remodel thenewly formed bone into stronger, more mature bone. The inhibition ofbone resorption would be expected to impair the later stages of normalfracture healing. Koivukangas et al., Long-term administration ofclodronate does not prevent fracture healing in rats. ClinicalOrthopaedics and Related Research 408: 268-278 (2003) and Peter et al.Effect of alendronate on fracture healing and bone remodeling in dogs.Journal of Orthopaedic Research 14: 74-79 (1996) disclose the effects ofbisphosphonate therapy on fracture healing. The data show thatbisphosphonate therapy which impairs osteoclast activity and boneremodeling does not inhibit the initial stages of fracture repair butdoes impair the later bone remodeling stage. The bisphosphonate effecton fracture healing reveals itself as persistence of a large fracturecallus that contains mechanically immature, woven bone rather thanmechanically mature, lamellar bone.

WO 03/066048 discloses that 12/15-lipoxygenase inhibitors can be used toprevent bone loss or increase bone mass. The publication describes datashowing that bone mineral density is preserved in transgenic mouse thatoverexpress IL-4 and that were treated with a 15-LO inhibitor. Thepublication does not disclose that 15-LO inhibitors can aid fracturehealing or the treatment of non-unions.

Traianedes, K., et al., 5-Lipoxygenase metabolites inhibit boneformation in vitro. Endocrinology, 139: 3178-3184 (1998) discloses theeffects of LTB4,5-HETE, and LTD4 (all products of 5-LO function) on thedifferentiation of fetal rat calvaria (osteoblast) cells. The data showthat 5-HETE and LTB4 reduce bone nodule formation and alkalinephosphatase activity in vitro but that LTD4 had no effect. The resultsfrom an in vitro organ culture model showed that LTB4 or 5-HETEtreatment prevented a BMP2 induced increase in mouse calvaria thickness.The publication, however, does not disclose the use of any 5-LOinhibitors, nor does it disclose that 5-LO inhibition would lead to thesame effect in cultured osteoblasts or in organ cultures. Similarly, Renand Dziak, Effects of leukotrienes on osteoblast cell proliferation.Calcified Tissue International 49: 197-201 (1991) discloses that LTB4treatment reduces proliferation of primary rat calvaria (osteoblast)cultures in vitro, but that LTB4 can promote proliferation ofestablished osteoblast cell lines (Saos-2 and G292) in vitro at higherconcentration (0.3-1 micromolar). Ren and Dziak also disclose that LTC4had no effect on the proliferation of primary rat osteoblast cells orSaos-2 cells but did promote proliferation of G292 cells. Further, Renand Dziak disclose that treatment of Saos-2 cells with a 5-LO inhibitor(AA-861) had no effect on Saos-2 cell proliferation. The publicationindicates that 5-LO inhibition should have no effect on osteogenesis.

Thus, it is readily apparent that compositions and methods foraccelerating or enhancing bone formation or fracture healing would behighly desirable.

SUMMARY

The present invention provides methods of promoting osteogenesis byadministering a compound that reduces a 5-lipoxygenase activity to treata bone fracture, a bone defect or a condition treated by inducing boneformation.

In another aspect of the invention, the methods can further comprise anadditional active agent such as a modulator of the activity of acyclooxygenase. In one aspect the activity of a cyclooxygenase-2 (COX-2)is increased. In another aspect, the activity of cyclooxygenase-1(COX-1) is reduced.

In one aspect, the methods use in vivo administration of a compound. Inanother aspect, ex vivo administration of a compound is used.

In one aspect, the compound is a small molecule. In another aspect thecompound is an antisense compound. In another aspect, the compound is anRNAi compound.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attachedfigures. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions, and aretherefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 summarizes an exemplary arachidonic acid metabolic or signalingpathway.

FIG. 2 illustrates the modulation of arachidonic acid metabolism byaltering cyclooxygenase activity or lipoxygenase activity to accelerateor enhance bone formation. FIG. 2A represents the normal functioning ofthe pathway. FIG. 2B shows that the inhibition of COX-2 activity leadsto excess leukotriene production which impairs bone formation infracture healing or other osteogenic processes. FIG. 2C shows that theinhibition of lipoxygenase activity leads to excess prostaglandinproduction which accelerates or enhances bone formation in fracturerepair or other osteogenic processes.

FIG. 3 shows that serial x-rays of femur fractures made from a 5LO−/−mouse and a normal mouse (C57BL/6). The x-rays show that osteogenesis,and therefore fracture healing is accelerated in the 5LO−/− mouse.

FIG. 4 illustrates mechanical testing data of fracture healing inwild-type (WT) and 5-LO knockout mice (5LO-KO or 5-LO−/−) 28 days and 84days after the onset of the fracture. Peak torque (FIG. 4A), rigidity(FIG. 4B), maximum shear stress (FIG. 4C), and shear modulus (FIG. 4D)were calculated from callus dimensions and the torque to angulardisplacement curves.

FIG. 5 illustrates histomorphometric data of fracture healing fromwild-type (WT) and 5-LO knockout mice (5-LOKO or 5-LO−/−) at 7, 10, 14,and 21 days after fracture. The left panel shows the percent of fracturecallus area that is newly formed bone (mineralized tissue) and the rightpanel shows the percent of fracture callus area that is cartilage.

FIG. 6 shows that fracture healing is dramatically impaired in COX-2knock-out mice and that the defect in healing occurs because of lack ofosteogenesis (new bone formation). FIG. 6A shows data from x-rays andFIGS. 6B and 6C show the histological samples of 14-day old femurfractures in mice lacking a functional COX-1 gene. FIG. 6D shows datafrom x-rays and FIGS. 6E and 6F show the histological samples of 14-dayold femur fractures in mice lacking a functional COX-2 gene.

FIG. 7 illustrates that osteogenesis is accelerated in rats treated with5-LO inhibitors, resulting in fractures healing faster than in untreatedrats.

FIG. 8 illustrates that osteogenesis is accelerated in rats treated withtwo different 5-LO inhibitors, resulting in fractures healing fasterthan in untreated rats. FIGS. 8A, 8B, and 8C show data from x-rays forvehicle control (8A), NDGA (8B), and AA-861 (8C). FIG. 8D is a graphshowing inhibition of 5-LO increases fracture callus peak torque.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

I. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

By “modulating an arachidonic acid metabolic or signaling pathway” ismeant use of a drug or a compound which inhibits or promotes theactivity or concentration of any enzyme or regulatory molecule involvedin an arachidonic acid metabolism or signal pathway in a cell or animal.Preferably drug or a compound can be selected from a FLAP inhibitor suchas BAYx 1005, MK-886, and MK-0591; a 5-Lipoxygenase inhibitor such asZileuton, BAY-G576, RS-43,179, Wy-47,288, ABT-761, vitamin A, and BWA4C; leukotriene receptor antagonists such as zafirlukast, montelukast,pranlukast, ICI-204,219, MK-571, MK-679, ONO-RS-411, SK&F 104,353, andWy-48,252; a leukotriene B4 receptor antagonists; a leukotriene C4synthase inhibitors; a Leukotriene A4 hydrolase inhibitors; anon-steroidal antiinflammatory drug (NSAID), a leukotriene receptorantagonists and leukotriene analogs, compounds modulating the formationand action of leukotrienes, compounds that affect cyclooxygenaseactivity, compounds that affect prostaglandin activity such as receptoragonists or antagonists, prostaglandin analogs, compounds that affectleukotriene activity such as receptor agonists or antagonists, andleukotriene analogs.

By “accelerated” is meant that osteogenesis occurs more rapidly and thetime required for bone healing is reduced, or the bone heals morequickly in a treated subject as compared to an untreated subject or acontrol subject.

By “enhancing” is meant that the healed bone in the treated subject hasimproved characteristics compared to an untreated subject, or a controlsubject such as, for example, greater bone strength.

By “fracture healing” or “fracture repair” is meant that, in particular,promoting the healing of bone fractures and bone defects, and improvingthe mechanical stability of the healing fracture or site. Such bonefractures may be, for example, the common, traumatic (disabling andnon-osteoporotic) fractures, the osteoporotic fractures due toosteoporosis or osteopenia of any etiology, fractures due to Paget'sdisease or fractures due to bone loss as a consequence of side effectsof other drugs, e.g. in patients receiving high doses ofcorticosteroids, fractures arising from other congenital or acquireddisease such as, e.g., osteogenesis imperfecta and breast cancer,surgical created fractures (osteotomies) used for example in bonelengthening and limb lengthening procedures, and treatment of bonefracture delayed unions or non-unions. The invention augments fracturehealing following normal reduction and immobilization of the fractureusing techniques common to one skilled in the art by accelerating andenhancing bone formation.

By “bone formation” is meant that the rate of bone formation in asubject treated according to the methods of the invention, such as,e.g., by receiving a 5-lipoxygenase inhibitor, is increased over thebone formation rate in a subject that is not given a 5-lipoxygenaseinhibitor. Such enhanced bone formation is determined herein using,e.g., quantitative digitized morphometry, as well as by other markers ofbone formation, as described above. Bone formation is meant to includethe osteogenic process used for spine fusions and other joint or boneankylosis application, bone formation into or around prosthetic devices,or bone formation to augment existing bones or replace missing bones orbone segments.

By “osteogenesis” is meant the production of bone that is associatedwith repair of a fractured bone, repair of a bone that has a defectcaused by intentional or non-intentional damage, or induction of boneformation used to fuse more than one bone or bone segment together.“Osteogenesis” is not meant to include bone formation associated withnormal bone growth in adolescents. “Osteogenesis” also is not meant toinclude bone formation associated with normal bone homeostasis, which isoften referred to as bone remodeling, in which bone is normallyturned-over by a process whereby osteoclasts resorb bone and osteoblastsmake new bone to replace that which has been resorbed.

By “bone defect” is meant damage to a bone such that a portion of thebone is removed or is otherwise missing. Such bone defects would includeanomalous holes, gaps or openings created in the bone for purposes of adiagnostic or therapeutic procedure, loss of bone segments from traumaor disease, puncture wounds to the bone, and the like.

The term “modulating” refers to the effect of a modulator on anarachidonic acid metabolic or signaling pathway. A modulator can be,e.g., a polypeptide, nucleic acid, macromolecule, complex molecule,small molecule, compound, or the like (naturally occurring ornon-naturally occurring) that is capable of causing modulation.Modulators can be evaluated for potential activity as inhibitors oractivators (directly or indirectly) of a functional property, biologicalactivity or process, or a combination thereof (e.g., agonist, partialantagonist, partial agonist, inverse agonist, antagonist, and the like),by inclusion in assays that measure the activity of an enzyme in thepathway.

The terms “effective amount” or “pharmaceutically effective amount”refer to a sufficient amount of an agent to provide the desiredbiological result. That result can be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. For example, an “effective amount”for therapeutic uses is the amount of the composition comprising anactive compound herein required to provide a clinically significantincrease in osteogenesis and, thus, healing rates in fracture repair;reversal of cartilage defects or disorders; stimulation and/oraugmentation of bone formation in fracture non-unions, delayed unionsand distraction osteogenesis; increase and/or acceleration of bonegrowth into prosthetic devices; enhanced or accelerated bone formationin joint ankylosis, bone ankylosis, or spine fusions, bone formation toaugment existing bone or replace missing bone or bone segments such asduring autograft, allograft, or synthetic bone material incorporation,and repair of dental defects.

As used herein, the terms “treat” or “treatment” are usedinterchangeably and are meant to indicate administering one or morecompounds in accordance with the methods of the invention to promoteosteogenesis to obtain a desired therapeutic objective. The termsfurther include ameliorating existing bone or cartilage deficitsymptoms, preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, and/or encouraging bone growth.

As used herein, “small molecule” is meant to indicate a chemicalcompound having a molecular weight of less than about 500 daltons. Smallmolecules do not include biologic polymers such as polypeptides andpolynucleotides.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual without causingany undesirable biological effects or interacting in a deleteriousmanner with any of the components of the composition in which it iscontained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.2 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, the term “subject” encompasses mammals. Examples ofmammals include, but are not limited to, any member of the Mammaliaclass: humans, non-human primates such as chimpanzees, and other apesand monkey species; farm animals such as cattle, horses, sheep, goats,swine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice and guinea pigs, and thelike. The term does not denote a particular age or gender.

The compounds of the present invention may be used to inhibit or reducethe activity of 5-lipoxygenase, 5-lipoxygenase and cyclooxygenase, andother enzymes and compounds in an arachadonic acid metabolic orsignaling pathway. In this context, inhibition and reduction of theenzyme activity refers to a lower level of measured activity relative toa control experiment in which the enzyme, cell, or subject is nottreated with the test compound. In particular embodiments, theinhibition or reduction in the measured activity is at least a 10%reduction or inhibition. One of skill in the art will appreciate thatreduction or inhibition of the measured activity of at least 20%, 50%,75%, 90% or 100% or any amount between 10% and 100%, may be preferredfor particular applications. Inhibition of enzyme activity may bethrough any mechanism, including, by way of example, but not limitation,a reduction in the amount of enzyme present, a competitive ornon-competitive inhibition of catalytic activity, an interference withan interaction between the enzyme and a co-factor or accessory protein,etc. In addition, the compounds of the present invention may be used toincrease a COX-2 activity. In particular embodiments, the increase ofenzyme activity refers to a higher level of measured activity relativeto a control experiment in which the enzyme, cell, or subject is nottreated with the test compound. In particular embodiments, the increasein measured activity is at least a 10% increase. One of skill in the artwill appreciate that an increase of the measured activity of at least20%, 50%, 75%, 90% or 100% or any amount between 10% and 100% or beyond,may be preferred for particular applications. Increase of enzymeactivity may be through any mechanism, including, by way of example butnot limitation, an increase in the amount of enzyme present, or byincreasing the enzyme's turnover rate, or altering its substrate bindingproperties.

References to the enzymes 5-lipoxygenase (5-LO), COX-1, and COX-2 areintended to encompass the exemplary sequences referenced in Table 1,some of which are provided immediately following Table 1, as well assequences at least 90% identical, or at least 95%, or at least 96%, orat least 97%, or at least 98%, or at least 99% identical to theexemplary sequences as can be ascertained by one of ordinary skill usingroutine alignment algorithms such as e.g., BLAST. In addition, othermammalian homologues are encompassed. Such homologues are identified assuch on the basis of e.g., sequence similarity, functional similarity,and by chromosome location. In addition to protein sequence, exemplarynucleic acid sequences are provided from which one of ordinary skill canreadily obtain sequences of anti-sense and RNAi compounds useful forinhibiting the activity of the enzyme in accordance with the methods ofthe invention. Anti-sense compounds useful for practice of the inventionare known in the art and can be obtained through commercial sources, asdescribed in, e.g., Ding et al. (1999) BBRC Vol. 261, pp. 218-223(incorporated by reference).

TABLE 1 Exemplary Sequences Entrez GeneBank Protein Similarity OMIM GeneAccession mRNA (Swiss- to human Symbol ID ID Number (GenBank) Prot)sequence Name: arachidonate 5-lipoxygenase; aka: 5-LO, 5-lipoxygenaseHuman ALOX5 152390   240 NC_000010 NM_000698 P09917 NA Rat Alox5 NA25290 NC_005103 NM_012822 P12527 86.29% (n¹) 92.94% (p²) Mouse Alox5 NA11689 NC_000072 NM_009662 P48999 87.88% (n) 93.47% (p)Name: arachidonate 5-lipoxygenase-activating protein; aka: FLAP HumanALOX5AP 603700   241 NC_000013 NM_001629 P20292 NA Rat Alox5ap NA 29624NC_005111 NM_017260 P20291 85.09% (n) 91.93% (p) Mouse Alox5ap NA 11690NC_000071 NM_009663 P30355 85.71% (n) 91.93% (p)Name: prostaglandin-endoperoxide synthase 2; aka: cyclooxygenase-2, COX-2,PGHS-2 Human PTGS2 600262  5743 NC_000001 NM_000963 P35354 NA Rat Ptgs2NA 29527 NC_005112 NM_017232 P35355 83.17% (n) 84.91% (p) Mouse Ptgs2 NA19225 NC_000067 NM_011198 Q05769 84.71% (n) 86.75% (p)Name: prostaglandin-endoperoxide synthase 1; aka: cyclooxygenase-1, COX-1,PGHS-1 Human PTGS1 176805  5742 NC_000009 NM_000962 P23219 NA Rat Ptgs1NA 24693 NC_005102 NM_017043 Q63921 84.87% (n) 88.11% (p) Mouse Ptgsl NA19224 NC_000068 NM_008969 P22437 85.59% (n) 89.78% (p)Human 5-Lipoxygenase mRNA Sequence (GenBank RefSeq NM_000698)(SEQ ID NO: 1)    1gccagggacc agtggtggga ggaggctgcg gcgctagatg cggacacctg gaccgccgcg   61ccgaggctcc cggcgctcgc tgctcccgcg gcccgcgcca tgccctccta cacggtcacc  121gtggccactg gcagccagtg gttcgccggc actgacgact acatctacct cagcctcgtg  181ggctcggcgg gctgcagcga gaagcacctg ctggacaagc ccttctacaa cgacttcgag  241cgtggcgcgg tggattcata cgacgtgact gtggacgagg aactgggcga gatccagctg  301gtcagaatcg agaagcgcaa gtactggctg aatgacgact ggtacctgaa gtacatcacg  361ctgaagacgc cccacgggga ctacatcgag ttcccctgct accgctggat caccggcgat  421gtcgaggttg tcctgaggga tggacgcgca aagttggccc gagatgacca aattcacatt  481ctcaagcaac accgacgtaa agaactggaa acacggcaaa aacaatatcg atggatggag  541tggaaccctg gcttcccctt gagcatcgat gccaaatgcc acaaggattt accccgtgat  601atccagtttg atagtgaaaa aggagtggac tttgttctga attactccaa agcgatggag  661aacctgttca tcaaccgctt catgcacatg ttccagtctt cttggaatga cttcgccgac  721tttgagaaaa tctttgtcaa gatcagcaac actatttctg agcgggtcat gaatcactgg  781caggaagacc tgatgtttgg ctaccagttc ctgaatggct gcaaccctgt gttgatccgg  841cgctgcacag agctgcccga gaagctcccg gtgaccacgg agatggtaga gtgcagcctg  901gagcggcagc tcagcttgga gcaggaggtc cagcaaggga acattttcat cgtggacttt  961gagctgctgg atggcatcga tgccaacaaa acagacccct gcacactcca gttcctggcc 1021gctcccatct gcttgctgta taagaacctg gccaacaaga ttgtccccat tgccatccag 1081ctcaaccaaa tcccgggaga tgagaaccct attttcctcc cttcggatgc aaaatacgac 1141tggcttttgg ccaaaatctg ggtgcgttcc agtgacttcc acgtccacca gaccatcacc 1201caccttctgc gaacacatct ggtgtctgag gtttttggca ttgcaatgta ccgccagctg 1261cctgctgtgc accccatttt caagctgctg gtggcacacg tgagattcac cattgcaatc 1321aacaccaagg cccgtgagca gctcatctgc gagtgtggcc tctttgacaa ggccaacgcc 1381acagggggcg gtgggcacgt gcagatggtg cagagggcca tgaaggacct gacctatgcc 1441tccctgtgct ttcccgaggc catcaaggcc cggggcatgg agagcaaaga agacatcccc 1501tactacttct accgggacga cgggctcctg gtgtgggaag ccatcaggac gttcacggcc 1561gaggtggtag acatctacta cgagggcgac caggtggtgg aggaggaccc ggagctgcag 1621gacttcgtga acgatgtcta cgtgtacggc atgcggggcc gcaagtcctc aggcttcccc 1681aagtcggtca agagccggga gcagctgtcg gagtacctga ccgtggtgat cttcaccgcc 1741tccgcccagc acgccgcggt caacttcggc cagtacgact ggtgctcctg gatccccaat 1801gcgcccccaa ccatgcgagc cccgccaccg actgccaagg gcgtggtgac cattgagcag 1861atcgtggaca cgctgcccga ccgcggccgc tcctgctggc atctgggtgc agtgtgggcg 1921ctgagccagt tccaggaaaa cgagctgttc ctgggcatgt acccagaaga gcattttatc 1981gagaagcctg tgaaggaagc catggcccga ttccgcaaga acctcgaggc cattgtcagc 2041gtgattgctg agcgcaacaa gaagaagcag ctgccatatt actacttgtc cccagaccgg 2101attccgaaca gtgtggccat ctgagcacac tgccagtctc actgtgggaa ggccagctgc 2161cccagccaga tggactccag cctgcctggc aggctgtctg gccaggcctc ttggcagtca 2221catctcttcc tccgaggcca gtacctttcc atttattctt tgatcttcag ggaactgcat 2281agattgatca aagtgtaaac accataggga cccattctac acagagcagg actgcacagc 2341gtcctgtcca cacccagctc agcatttcca caccaagcag caacagcaaa tcacgaccac 2401tgatagatgt ctattcttgt tggagacatg ggatgattat tttctgttct atttgtgctt 2461agtccaattc cttgcacata gtaggtaccc aattcaatta ctattgaatg aattaagaat 2521tggttgccat aaaaataaat cagttcattt aaaaaaaaaa aaaaaaaaHuman 5-Lipoxygenase Protein Sequence (GenBank RefSeq NM_000698)(SEQ ID NO: 2)MPSYTVTVATGSQWFAGTDDYIYLSLVGSAGCSEKHLLDKPFYNDFERGAVDSYDVTVDEELGEIQLVRIEKRKYWLNDDWYLKYITLKTPHGDYIEFPCYRWITGDVEVVLRDGRAKLARDDQIHILKQHRRKELETRQKQYRWMEWNPGFPLSIDAKCHKDLPRDIQFDSEKGVDFVLNYSKAMENLFINRFMHMFQSSWNDFADFEKIFVKISNTISERVMNHWQEDLMFGYQFLNGCNPVLIRRCTELPEKLPVTTEMVECSLERQLSLEQEVQQGNIFIVDFELLDGIDANKTDPCTLQFLAAPICLLYKNLANKIVPIAIQLNQIPGDENPIFLPSDAKYDWLLAKIWVRSSDFHVHQTITHLLRTHLVSEVFGIAMYRQLPAVHPIFKLLVAHVRFTIAINTKAREQLICECGLFDKANATGGGGHVQMVQRAMKDLTYASLCFPEAIKARGMESKEDIPYYFYRDDGLLVWEAIRTFTAEVVDIYYEGDQVVEEDPELQDFVNDVYVYGMRGRKSSGFPKSVKSREQLSEYLTVVIFTASAQHAAVNFGQYDWCSWIPNAPPTMRAPPPTAKGVVTIEQIVDTLPDRGRSCWHLGAVWALSQFQENELFLGMYPEEHFIEKPVKEAMARFRKNLEAIVSVIAERNKKKQLPYYYLSPDRIPNSVAIHuman FLAP mRNA Sequence (GenBank RefSeq NM_001629) (SEQ ID NO: 3)    1acttcccctt cctgtacagg gcaggttgtg cagctggagg cagagcagtc ctctctgggg   61agcctgaagc aaacatggat caagaaactg taggcaatgt tgtcctgttg gccatcgtca  121ccctcatcag cgtggtccag aatggattct ttgcccataa agtggagcac gaaagcagga  181cccagaatgg gaggagcttc cagaggaccg gaacacttgc ctttgagcgg gtctacactg  241ccaaccagaa ctgtgtagat gcgtacccca ctttcctcgc tgtgctctgg tctgcggggc  301tactttgcag ccaagttcct gctgcgtttg ctggactgat gtacttgttt gtgaggcaaa  361agtactttgt cggttaccta ggagagagaa cgcagagcac ccctggctac atatttggga  421aacgcatcat actcttcctg ttcctcatgt ccgttgctgg catattcaac tattacctca  481tcttcttttt cggaagtgac tttgaaaact acataaagac gatctccacc accatctccc  541ctctacttct cattccctaa ctctctgctg aatatggggt tggtgttctc atctaatcaa  601tacctacaag tcatcataat tcagctcttg agagcattct gctcttcttt agatggctgt  661aaatctattg gccatctggg cttcacagct tgagttaacc ttgcttttcc gggaacaaaa  721tgatgtcatg tcagctccgc cccttgaaca tgaccgtggc cccaaatttg ctattcccat  781gcattttgtt tgtttcttca cttatcctgt tctctgaaga tgttttgtga ccaggtttgt  841gttttcttaa aataaaatgc agagacatgt tttHuman FLAP Protein Sequence (GenBank RefSeq NM_001629) (SEQ ID NO: 4)MDQETVGNVVLLAIVTLISVVQNGFFAHKVEHESRTQNGRSFQRTGTLAFERVYTANQNCVDAYPTFLAVLWSAGLLCSQVPAAFAGLMYLFVRQKYFVGYLGERTQSTPGYIFGKRIILFLFLMSVAGIFNYYLIFFFGSDFENYIKTISTTISPLLLIP Human COX-2 mRNA Sequence (GenBank RefSeq NM_00963)(SEQ ID NO: 5)    1caattgtcat acgacttgca gtgagcgtca ggagcacgtc caggaactcc tcagcagcgc   61ctccttcagc tccacagcca gacgccctca gacagcaaag cctacccccg cgccgcgccc  121tgcccgccgc tcggatgctc gcccgcgccc tgctgctgtg cgcggtcctg gcgctcagcc  181atacagcaaa tccttgctgt tcccacccat gtcaaaaccg aggtgtatgt atgagtgtgg  241gatttgacca gtataagtgc gattgtaccc ggacaggatt ctatggagaa aactgctcaa  301caccggaatt tttgacaaga ataaaattat ttctgaaacc cactccaaac acagtgcact  361acatacttac ccacttcaag ggattttgga acgttgtgaa taacattccc ttccttcgaa  421atgcaattat gagttatgtc ttgacatcca gatcacattt gattgacagt ccaccaactt  481acaatgctga ctatggctac aaaagctggg aagccttctc taacctctcc tattatacta  541gagcccttcc tcctgtgcct gatgattgcc cgactccctt gggtgtcaaa ggtaaaaagc  601agcttcctga ttcaaatgag attgtggaaa aattgcttct aagaagaaag ttcatccctg  661atccccaggg ctcaaacatg atgtttgcat tctttgccca gcacttcacg catcagtttt  721tcaagacaga tcataagcga gggccagctt tcaccaacgg gctgggccat ggggtggact  781taaatcatat ttacggtgaa actctggcta gacagcgtaa actgcgcctt ttcaaggatg  841gaaaaatgaa atatcagata attgatggag agatgtatcc tcccacagtc aaagatactc  901aggcagagat gatctaccct cctcaagtcc ctgagcatct acggtttgct gtggggcagg  961aggtctttgg tctggtgcct ggtctgatga tgtatgccac aatctggctg cgggaacaca 1021acagagtatg cgatgtgctt aaacaggagc atcctgaatg gggtgatgag cagttgttcc 1081agacaagcag gctaatactg ataggagaga ctattaagat tgtgattgaa gattatgtgc 1141aacacttgag tggctatcac ttcaaactga aatttgaccc agaactactt ttcaacaaac 1201aattccagta ccaaaatcgt attgctgctg aatttaacac cctctatcac tggcatcccc 1261ttctgcctga cacctttcaa attcatgacc agaaatacaa ctatcaacag tttatctaca 1321acaactctat attgctggaa catggaatta cccagtttgt tgaatcattc accaggcaaa 1381ttgctggcag ggttgctggt ggtaggaatg ttccacccgc agtacagaaa gtatcacagg 1441cttccattga ccagagcagg cagatgaaat accagtcttt taatgagtac cgcaaacgct 1501ttatgctgaa gccctatgaa tcatttgaag aacttacagg agaaaaggaa atgtctgcag 1561agttggaagc actctatggt gacatcgatg ctgtggagct gtatcctgcc cttctggtag 1621aaaagcctcg gccagatgcc atctttggtg aaaccatggt agaagttgga gcaccattct 1681ccttgaaagg acttatgggt aatgttatat gttctcctgc ctactggaag ccaagcactt 1741ttggtggaga agtgggtttt caaatcatca acactgcctc aattcagtct ctcatctgca 1801ataacgtgaa gggctgtccc tttacttcat tcagtgttcc agatccagag ctcattaaaa 1861cagtcaccat caatgcaagt tcttcccgct ccggactaga tgatatcaat cccacagtac 1921tactaaaaga acgttcgact gaactgtaga agtctaatga tcatatttat ttatttatat 1981gaaccatgtc tattaattta attatttaat aatatttata ttaaactcct tatgttactt 2041aacatcttct gtaacagaag tcagtactcc tgttgcggag aaaggagtca tacttgtgaa 2101gacttttatg tcactactct aaagattttg ctgttgctgt taagtttgga aaacagtttt 2161tattctgttt tataaaccag agagaaatga gttttgacgt ctttttactt gaatttcaac 2221ttatattata agaacgaaag taaagatgtt tgaatactta aacactatca caagatggca 2281aaatgctgaa agtttttaca ctgtcgatgt ttccaatgca tcttccatga tgcattagaa 2341gtaactaatg tttgaaattt taaagtactt ttggttattt ttctgtcatc aaacaaaaac 2401aggtatcagt gcattattaa atgaatattt aaattagaca ttaccagtaa tttcatgtct 2461actttttaaa atcagcaatg aaacaataat ttgaaatttc taaattcata gggtagaatc 2521acctgtaaaa gcttgtttga tttcttaaag ttattaaact tgtacatata ccaaaaagaa 2581gctgtcttgg atttaaatct gtaaaatcag atgaaatttt actacaattg cttgttaaaa 2641tattttataa gtgatgttcc tttttcacca agagtataaa cctttttagt gtgactgtta 2701aaacttcctt ttaaatcaaa atgccaaatt tattaaggtg gtggagccac tgcagtgtta 2761tctcaaaata agaatatttt gttgagatat tccagaattt gtttatatgg ctggtaacat 2821gtaaaatcta tatcagcaaa agggtctacc tttaaaataa gcaataacaa agaagaaaac 2881caaattattg ttcaaattta ggtttaaact tttgaagcaa actttttttt atccttgtgc 2941actgcaggcc tggtactcag attttgctat gaggttaatg aagtaccaag ctgtgcttga 3001ataacgatat gttttctcag attttctgtt gtacagttta atttagcagt ccatatcaca 3061ttgcaaaagt agcaatgacc tcataaaata cctcttcaaa atgcttaaat tcatttcaca 3121cattaatttt atctcagtct tgaagccaat tcagtaggtg cattggaatc aagcctggct 3181acctgcatgc tgttcctttt cttttcttct tttagccatt ttgctaagag acacagtctt 3241ctcatcactt cgtttctcct attttgtttt actagtttta agatcagagt tcactttctt 3301tggactctgc ctatattttc ttacctgaac ttttgcaagt tttcaggtaa acctcagctc 3361aggactgcta tttagctcct cttaagaaga ttaaaagaga aaaaaaaagg cccttttaaa 3421aatagtatac acttatttta agtgaaaagc agagaatttt atttatagct aattttagct 3481atctgtaacc aagatggatg caaagaggct agtgcctcag agagaactgt acggggtttg 3541tgactggaaa aagttacgtt cccattctaa ttaatgccct ttcttattta aaaacaaaac 3601caaatgatat ctaagtagtt ctcagcaata ataataatga cgataatact tcttttccac 3661atctcattgt cactgacatt taatggtact gtatattact taatttattg aagattatta 3721tttatgtctt attaggacac tatggttata aactgtgttt aagcctacaa tcattgattt 3781ttttttgtta tgtcacaatc agtatatttt ctttggggtt acctctctga atattatgta 3841aacaatccaa agaaatgatt gtattaagat ttgtgaataa atttttagaa atctgattgg 3901catattgaga tatttaaggt tgaatgtttg tccttaggat aggcctatgt gctagcccac 3961aaagaatatt gtctcattag cctgaatgtg ccataagact gaccttttaa aatgttttga 4021gggatctgtg gatgcttcgt taatttgttc agccacaatt tattgagaaa atattctgtg 4081tcaagcactg tgggttttaa tatttttaaa tcaaacgctg attacagata atagtattta 4141tataaataat tgaaaaaaat tttcttttgg gaagagggag aaaatgaaat aaatatcatt 4201aaagataact caggagaatc ttctttacaa ttttacgttt agaatgttta aggttaagaa 4261agaaatagtc aatatgcttg tataaaacac tgttcactgt tttttttaaa aaaaaaactt 4321gatttgttat taacattgat ctgctgacaa aacctgggaa tttgggttgt gtatgcgaat 4381gtttcagtgc ctcagacaaa tgtgtattta acttatgtaa aagataagtc tggaaataaa 4441tgtctgttta tttttgtact atttaHuman COX-2 Protein Sequence (GenBank RefSeq NM_000963) (SEQ ID NO: 6)MLARALLLCAVLALSHTANPCCSHPCQNRGVCMSVGFDQYKCDCTRTGFYGENCSTPEFLTRIKLFLKPTPNTVHYILTHFKGFWNVVNNIPFLRNAIMSYVLTSRSHLIDSPPTYNADYGYKSWEAFSNLSYYTRALPPVPDDCPTPLGVKGKKQLPDSNEIVEKLLLRRKFIPDPQGSNMMFAFFAQHFTHQFFKTDHKRGPAFTNGLGHGVDLNHIYGETLARQRKLRLFKDGKMKYQIIDGEMYPPTVKDTQAEMIYPPQVPEHLRFAVGQEVFGLVPGLMMYATIWLREHNRVCDVLKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFNKQFQYQNRIAAEFNTLYHWHPLLPDTFQIHDQKYNYQQFIYNNSILLEHGITQFVESFTRQIAGRVAGGRNVPPAVQKVSQASIDQSRQMKYQSFNEYRKRFMLKPYESFEELTGEKEMSAELEALYGDIDAVELYPALLVEKPRPDAIFGETMVEVGAPFSLKGLMGNVICSPAYWKPSTFGGEVGFQIINTASIQSLICNNVKGCPFTSFSVPDPELIKTVTINASSSRSGLDDINPTVLLKERSTEL Human COX-1 mRNA Sequence (GenBank RefSeq NM_000962) (SEQ ID NO: 7)   1 aggtgacagc tggagggagg agcgggggtg gagccggggg aagggtgggg aggggatggg  61 ctggagctcc gggcagtgtg cgaggcgcac gcacaggagc ctgcactctg cgtcccgcac 121 cccagcagcc gcgccatgag ccggagtctc ttgctctggt tcttgctgtt cctgctcctg 181 ctcccgccgc tccccgtcct gctcgcggac ccaggggcgc ccacgccagt gaatccctgt 241 tgttactatc catgccagca ccagggcatc tgtgtccgct tcggccttga ccgctaccag 301 tgtgactgca cccgcacggg ctattccggc cccaactgca ccatccctgg cctgtggacc 361 tggctccgga attcactgcg gcccagcccc tctttcaccc acttcctgct cactcacggg 421 cgctggttct gggagtttgt caatgccacc ttcatccgag agatgctcat gcgcctggta 481 ctcacagtgc gctccaacct tatccccagt ccccccacct acaactcagc acatgactac 541 atcagctggg agtctttctc caacgtgagc tattacactc gtattctgcc ctctgtgcct 601 aaagattgcc ccacacccat gggaaccaaa gggaagaagc agttgccaga tgcccagctc 661 ctggcccgcc gcttcctgct caggaggaag ttcatacctg acccccaagg caccaacctc 721 atgtttgcct tctttgcaca acacttcacc caccagttct tcaaaacttc tggcaagatg 781 ggtcctggct tcaccaaggc cttgggccat ggggtagacc tcggccacat ttatggagac 841 aatctggagc gtcagtatca actgcggctc tttaaggatg ggaaactcaa gtaccaggtg 901 ctggatggag aaatgtaccc gccctcggta gaagaggcgc ctgtgttgat gcactacccc 961 cgaggcatcc cgccccagag ccagatggct gtgggccagg aggtgtttgg gctgcttcct1021 gggctcatgc tgtatgccac gctctggcta cgtgagcaca accgtgtgtg tgacctgctg1081 aaggctgagc accccacctg gggcgatgag cagcttttcc agacgacccg cctcatcctc1141 ataggggaga ccatcaagat tgtcatcgag gagtacgtgc agcagctgag tggctatttc1201 ctgcagctga aatttgaccc agagctgctg ttcggtgtcc agttccaata ccgcaaccgc1261 attgccatgg agttcaacca tctctaccac tggcaccccc tcatgcctga ctccttcaag1321 gtgggctccc aggagtacag ctacgagcag ttcttgttca acacctccat gttggtggac1381 tatggggttg aggccctggt ggatgccttc tctcgccaga ttgctggccg gatcggtggg1441 ggcaggaaca tggaccacca catcctgcat gtggctgtgg atgtcatcag ggagtctcgg1501 gagatgcggc tgcagccctt caatgagtac cgcaagaggt ttggcatgaa accctacacc1561 tccttccagg agctcgtagg agagaaggag atggcagcag agttggagga attgtatgga1621 gacattgatg cgttggagtt ctaccctgga ctgcttcttg aaaagtgcca tccaaactct1681 atctttgggg agagtatgat agagattggg gctccctttt ccctcaaggg tctcctaggg1741 aatcccatct gttctccgga gtactggaag ccgagcacat ttggcggcga ggtgggcttt1801 aacattgtca agacggccac actgaagaag ctggtctgcc tcaacaccaa gacctgtccc1861 tacgtttcct tccgtgtgcc ggatgccagt caggatgatg ggcctgctgt ggagcgacca1921 tccacagagc tctgaggggc aggaaagcag cattctggag gggagagctt tgtgcttgtc1981 attccagagt gctgaggcca gggctgatgg tcttaaatgc tcattttctg gtttggcatg2041 gtgagtgttg gggttgacat ttagaacttt aagtctcacc cattatctgg aatattgtga2101 ttctgtttat tcttccagaa tgctgaactc cttgttagcc cttcagattg ttaggagtgg2161 ttctcatttg gtctgccaga atactgggtt cttagttgac aacctagaat gtcagatttc2221 tggttgattt gtaacacagt cattctagga tgtggagcta ctgatgaaat ctgctagaaa2281 gttagggggt tcttattttg cattccagaa tcttgacttt ctgattggtg attcaaagtg2341 ttgtgttcct ggctgatgat ccagaacagt ggctcgtatc ccaaatctgt cagcatctgg2401 ctgtctagaa tgtggatttg attcattttc ctgttcagtg agatatcata gagacggaga2461 tcctaaggtc caacaagaat gcattccctg aatctgtgcc tgcactgaga gggcaaggaa2521 gtggggtgtt cttcttggga cccccactaa gaccctggtc tgaggatgta gagagaacag2581 gtgggctgta ttcacgccat tggttggaag ctaccagagc tctatcccca tccaggtctt2641 gactcatggc agctgtttct catgaagcta ataaaattcg ctttctaaag ttacctgtta2701 tatatctctt ttggtcccat cctctaaagc agaggcaaca ctggaacatg gctagccttt2761 cttgtagcca tggctgggcg tgctagaggt tgcagcatga gactttctgc tgggatcctt2821 gggcccatca ctgtatagac atgctaccac tggtacttcc tttctccctg cgggccaggc2881 actgcccttt tcaggaagct ctcttaaaat acccattgcc ccagacctgg aagatataac2941 attcagttcc caccatctga ttaaaacaac ttcctccctt acagagcata caacagaggg3001 ggcacccggg gaggagagca catactgtgt tccaatttca cgcttttaat tctcatttgt3061 tctcacacca acagtgtgaa gtgcgtggta taatctccat ttcaaaacca aggaagcagc3121 ctcagagtgg tcgagtgaca cacctcacgc aggctgagtc cagagcttgt gctcctcttg3181 attcctggtt tgactcagtt ccaggcctga tcttgcctgt ctggctcagg gtcaaagaca3241 gaatggtgga gtgtagcctc cacctgatat tcaggctact cattcagtcc caaatatgta3301 ttttcctaag tgtttactat gtgccagttc ctgtaacagg tgtggggaca cagcagtgag3361 taatcaatac agacaaggtt ctgcccttat ggagctcaca ctccagtggc agacaaacag3421 accataaata aggaaacgat gaaataagat atatacaagg tgagtgtgac ttcccttcta3481 accccctctg ctctgtcctc ccctattgcg ctctcaagac cagagaccca acagcagtga3541 tctcagggca gacagccctc cactccagct ctgagaccct tttctcagga cctctgtagg3601 cagcagagag agaggacaga ggggtaagat gaggggttga gggaaggttc ttcatgatcc3661 acactttggg cttagtattt ctcaggaaga gctatggccc agaaacaaca ggggaaacta3721 gagttcggtc tgacagtcct tggggttaag tctcctgtct tatggtccag aaactcctgt3781 ttctccttag ttggctggaa actgctccca tcattccttc tggcctctgc tgaatgcagg3841 gaatgcaatc cttccctgct cttgcagttg ctctgacgta gaaagatcct tcgggtgctg3901 gaagtctcca tgaagagctt gtgtcctgtc ctttcttgca gattctattt cccctcttct3961 gctaatacct cttactttgc ttgagaatcc tctcctttct tattaatttc agtcttggtg4021 gttctatcag gggtgcattc tggccaaggg gtgggcctgt gaatcaatcc tgggcaatca4081 gacaccctct ccttaaaaac tggcccgtgg agactgagat cactgactct gactcatccc4141 cacagctggc tctgacaaga tggtccattt gttcctgctt ccgagatccc cagggcagcc4201 tggatccctg cccttctcaa gactttagct tttccttcca tccggtggcc tattccagga4261 attcctcttt tgcttaaatc agttggagtt tgtgtctgtt gcttgtaatc aagcctttat4321 ggctgctggg ctgagtgaca caagcacttt aatggcctgg agggactttt aatcagtgaa4381 gatgcaatca gacaagtgtt ttggaaagag caccctcgag aagggtggat gacagggcag4441 agcaggaagg acaggaagct ggcagaacgg aggaggctgc agccgtggtc caaccaggag4501 ctgatggcag ctggggctag gggaagggct ttgagggtgg aaggatggga tgggttccag4561 aggtattcct ctcttaaatg caagtgccta gattaggtag actttgctta gtattgacaa4621 ctgcacatga aagttttgca aagggaaaca ggctaaatgc accaagaaag cttcttcaga4681 gtgaagaatc ttaatgcttg taatttaaac atttgttcct ggagttttga tttggtggat4741 gtgatggttg gttttatttg tcagtttggt tgggctatag cacacagtta tttaatcaaa4801 cagtaatcta ggtgtggctg tgaaggtatt ttgtagatgt gattaacatc tacaatcagt4861 tgactttaag tgaaagagat tacttaaata atttgggtga gctgcacctg attagttgaa4921 aggcctcaag aacaaacact gcagtttcct ggaaaagaag aaactttgcc tcaagactat4981 agccatcgac tcctgcctga gtttccagcc tgctagtctg ccctatggat ttgaagtttg5041 ccaaccccaa caattgtgtg aattaatttc taaaaataaa gctatataca gccHuman COX-1 Protein Sequence (GenBank RefSeq NM_000962) (SEQ ID NO: 8)MSRSLLLWFLLFLLLLPPLPVLLADPGAPTPVNPCCYYPCQHQGICVRFGLDRYQCDCTRTGYSGPNCTIPGLWTWLRNSLRPSPSFTHFLLTHGRWFWEFVNATFIREMLMRLVLTVRSNLIPSPPTYNSAHDYISWESFSNVSYYTRILPSVPKDCPTPMGTKGKKQLPDAQLLARRFLLRRKFIPDPQGTNLMFAFFAQHFTHQFFKTSGKMGPGFTKALGHGVDLGHIYGDNLERQYQLRLFKDGKLKYQVLDGEMYPPSVEEAPVLMHYPRGIPPQSQMAVGQEVFGLLPGLMLYATLWLREHNRVCDLLKAEHPTWGDEQLFQTTRLILIGETIKIVIEEYVQQLSGYFLQLKFDPELLFGVQFQYRNRIAMEFNHLYHWHPLMPDSFKVGSQEYSYEQFLFNTSMLVDYGVEALVDAFSRQIAGRIGGGRNMDHHILHVAVDVIRESREMRLQPFNEYRKRFGMKPYTSFQELVGEKEMAAELEELYGDIDALEFYPGLLLEKCHPNSIFGESMIEIGAPFSLKGLLGNPICSPEYWKPSTFGGEVGFNIVKTATLKKLVCLNTKTCPYVSFRVPDASQDDGPAVERPSTEL¹Similarity between mRNA sequences. ²Similarity between proteinsequences.

II. 5-LIPOXYGENASE INHIBITORS

The applicant has discovered that inhibiting the activity of5-lipoxygenase promotes osteogenesis which can be used to accelerateand/or enhance the healing of a bone fracture, to treat a bone defect,or to treat by inducing bone formation. The applicant's discovery isbased on his hypothesis that a potential mechanism by which loss ofCOX-2 function could inhibit fracture healing was by shuntingarachidonic acid into the lipoxygenase pathway with consequent formationof abnormally high inhibitory 5-HETE, LTB4, or other 5-LO metabolitelevels (FIG. 2). During a normal inflammation response, such as afracture, the synthesis of prostaglandins and leukotrienes is balanced(FIG. 2A). Without being bound to a theory, the inventor theorizes thatinhibiting COX-2 function shunts arachidonic acid into the lipoxygenasepathway to produce excess leukotrienes thereby impairing bone formation(FIG. 2B). Conversely, by inhibiting 5-lipoxygenase activity,arachidonic acid is shunted into the cyclooxygenase pathway to produceexcess prostaglandins that accelerate or enhance bone formation (FIG.2C).

To test this potential mechanism, fracture healing was assessed in5-LO−/− mice. The applicant found that loss of 5-LO function accelerateshealing. Radiographic examination of fracture healing in age-matchedmice in the C57BL/6 background showed that fracture bridging occurred by2 weeks post-fracture in the 5-LO−/− mice as compared to 3 weekspost-fracture in the normal mice (FIG. 3). Further, callus remodelingwas significantly accelerated, thus the 5-LO−/− callus regains itsinitial structural and material properties much faster than in normalmice based upon torsional mechanical testing (FIG. 4 and TABLE 2). Thus,loss of 5-LO function accelerates and enhances fracture healing and boneformation.

Histological examination of calcified samples supported the radiographicdata. Plastic embedded, calcified sections of normal and 5-LO−/− mousefractures stained with Stevenel's blue and van Gieson's picrofuchsinshow that after just 2 weeks of healing the fracture was bridged withcalcified tissue in the 5-LO−/− mice while the normal mouse (C57BL/6)still had a cartilaginous soft callus. Histomorphometric measurements offracture callus cartilage area showed that cartilage area peaked by day7 post-fracture in 5-LO−/− mice and by day 10 post-fracture in normalmice (FIG. 5 and TABLE 3). Measurement of new bone (calcified tissue) inthe fracture callus showed that almost twice as much new bone in the5-LO−/− after 7 days of healing and significantly more new bone at day10 as well (FIG. 5 and TABLE 2). These data show that a normal, albeitsignificantly accelerated, endochondral ossification pathway is used toheal the fracture in the 5-LO−/− mice. Experiments using younger andolder 5-LO−/− mice and in different genetic backgrounds gave identicalresults: loss of 5-LO function results in accelerated bone regeneration.

The data from these experiments show that a 10 day fracture callus in5-LO−/− mouse is equivalent to a 14 day callus in a normal mouse; that a14 day 5-LO−/− callus is equivalent to a 21 day normal callus; and thata 1 month 5-LO−/− callus is equivalent to a 3 month normal callus (FIG.3). Thus, loss of 5-LO function accelerates and/or enhances theregenerative and remodeling phases of fracture healing.

In one aspect of the invention, compounds that inhibit 5-lipoxygenaseactivity accelerate and/or enhance healing of a bone fracture or preventbone resorption or promote bone formation provide important benefits toefforts at treating human disease. Compounds that inhibit 5-lipoxygenaseactivity can be used, e.g., in a method for treating bone fracture dueto trauma, or due to osteoporosis or osteoarthritis, in a method fortreating Paget's disease, in a method for treating other conditions suchas bone transplants and diseases associated with increased bonefracture, and in methods that require bone formation such as spinefusions, other bone and joint ankylosis procedures, bone or limblengthening, augmentation of bone structure, incorporation of allograft,autograft, or synthetic bone material into bone defects, bone growthinto or around prosthetic devices, and other similar procedures.

Several inhibitors of 5-lipoxygenase and their dosing are known whichare useful for practicing the methods of the invention. A 5-lipoxygenaseinhibitor can be3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoicacid (MK886) or derivatives thereof;3-(1-(4-chlorobenzyl)-3-(1-butyl-thio)-5-(quinolin-2-yl-methoxy)-indol-2-yl)-2,2-dimethylpropanoic acid) (MK-591) or derivatives thereof; nordihydroguaiareticacid (NDGA) or derivatives thereof;2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone(AA861) or derivatives thereof; or(N-(1-benzo(b)thien-2-ylethyl)-N-hydroxyurea) (Zileuton) or derivativesthereof. Derivatives include, e.g., pharmaceutically acceptable salts,prodrugs, etc. which also are useful as 5-lipoxygenase inhibitors.Derivatives of exemplary compounds are intended to be within the scopeof the claimed invention.

Other 5-lipoxygenase inhibitors for use in the invention includemasoprocol, tenidap, flobufen, lonapalene, tagorizine, Abbott A-121798,Abbott A-76745, Abbott A-78773, Abbott A-79175, Abbott ABT 761,Dainippon AL-3264, Bayer Bay-x-1005, Biofor BF-389, bunaprolast, CytomedCMI-392, Takeda CV-6504, enazadrem phosphate, Leo Denmark ETH-615,flezelastine hydrochloride, Merck Frosst L-663536, Merckle ML-3000, 3MPharmaceuticals R-840, rilopirox, Schering Plough SCH-40120, tepoxalin,linazolast (TMK-688), Zeneca ZD-2138, Bristol-Myers Squibb BU-4601A,carbazomycin C, lagunamycin, Wellcome BW-70C, Ciba-Geigy CGS-26529,Warner-Lambert CI 1004, Warner-Lambert PD-136005, Warner-LambertPD-145246, Elsai E-3040, Fujirebio F-1322, Fujisawa FR-110302, MerckFrosst L-699333, Merck Frosst L-739010, Lilly LY-269415, LillyLY-178002, Hoechst Roussel P-8892, SmithKline Beecham SB-202235,American Home Products WAY-121520, American Home Products WAY-125007,Zeneca ZD-7717, Zeneca ZM-216800, Zeneca ZM-230487,1,2-dihydro-n-(2-thiazolyl)-1-oxopyrrolo(3,2,1-kl)phenothiazine-1-carboxamide,Abbott A-65260, Abbott A-69412, Abbott-63162, American Home ProductsAHR-5333, Bayer Bay-q-1531, Boehringer Ingelheim BI-L-357, BoehringerIngelheim BI-L-93BS, Boehringer Ingelheim BIL 226XX, Bristol-MyersSquibb BMY-30094, carbazomycin B, Wellcome BW-B218C, Chauvin CBS-1114,Ciba-Geigy CGS-21595, Ciba-Geigy CGS-22745, Ciba-Geigy CGS-23885,Ciba-Geigy CGS 24891, Ciba-Geigy CGS-8515, Chiesi CHF-1909,Warner-Lambert CI-986, Warner-Lambert CI-987, cirsiliol, docebenone,Eisai E-5110, Eisai E-6080, enofelast, epocarbazolin-A, eprovafen,evandamine, Fisons FPL 62064, Zeneca ICI-211965, Zeneca ICI-216800,Kyowa Hakko KF-8940, Merck & Co L-651392, Merck & Co L-651896, Merck &Co L-652343, Merck & Co L-656224, Merck & Co L-670630, Merck & CoL-674636, Lilly LY-233569, Merck & Co MK-591, Merck & Co L-655240,nitrosoxacin-A, Ono ONO-5349, Ono ONO-LP-219, Ono ONO-LP-269,Warner-Lambert PD-127443, Purdue Frederick PF-5901, Rhone-Poulenc RorerRev-5367, Rhone-Poulenc Rorer RG-5901-A, Rhone-Poulenc Rorer RG-6866,Roussel-Uclaf RU-46057, Searle SC-41661A, Searle SC-45662, SandozSDZ-210-610, SmithKline Beecham SK&F-104351, SmithKline BeechamSK&F-104493, SmithKline Beecham SK&F-105809, Synthelabo SL-81-0433,Teijin TEI-8005, Terumo TMK-777, Terumo TMK-781, Terumo TMK-789, TerumoTMK-919, Terumo TMK-992, Teikoku Hormone TZI-41127, American HomeProducts WAY-120739, American Home Products WY-47288, American HomeProducts WY-48252, American Home Products WY-50295, Yoshitomi Y-19432,4-{3-[4-(2-methyl-1H-imidazol-1-yl)phenylthio]}-phenyl-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide,esculetin, phenidone and its derivatives, BI-L-239,5,8,11-eicosatriynoic acid (ETI), 5,8,11,14-eicosatetraynoic acid(ETYA), cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, curcumin,esculeitin, gossypol, caffeic acid, baicalein, 7,7-dimethyleicosadrenoicacid (DEDA), Ly311727, bromoenol lactone, methyl arachidonylfluorophosphonate, methyl γ-linolenyl fluorophosphonate, oleyoxyethylphosphorylcholine, AACOCF3, n-(p-amylcinnamoyl) anthranilic acid,mepacrine, quinacrine, atabrine, parabromophenacylbromide, aristolochicacid, corticosteroids, Glaxo SmithKline 480848, Glaxo SmithKline 659032,Glaxo SmithKline 677116, BMS-181162, MJ33, and MillenniumPharmaceuticals MLN977.

More preferred 5-lipoxygenase inhibitors include masoprocol, tenidap,zileuton, flobufen, lonapalene, tagorizine, Abbott A-121798, AbbottA-76745, Abbott A-78773,[(R)(+)N′-[[5-(4-fluorophenoxy)furan-2-yl]-1-methyl-2-propynyl]-N-hydroxyurea(Abbott A-79175),] Abbott A-79175, Abbott ABT 761, Dainippon AL-3264,Bayer Bay-x-1005, Biofor BF-389, bunaprolast, Cytomed CMI-392, TakedaCV-6504, Ciba-Geigy CGS-26529, enazadrem phosphate, Leo Denmark ETH-615,flezelastine hydrochloride, Merck Frosst L 663536, Merck Frosst L699333, Merckle ML-3000, 3M Pharmaceuticals R-840, rilopirox, ScheringPlough SCH 40120, tepoxalin, linazolast (TMK-688), Zeneca ZD-7717,Zeneca ZM-216800, Zeneca ZM-230487, Zeneca ZD-2138; and NDGA(nondihydroguaiaretic acid).

Even more preferred 5-lipoxygenase inhibitors include tenidap, zileuton,flobufen, lonapalene, tagorizine, AA-861, Abbott A-121798, AbbottA-76745, Abbott A-78773, Abbott A-79175, Abbott ABT 761, Ciba-GeigyCGS-26529, Biofor BF-389, Cytomed CMI-392, Leo Denmark ETH-615,lonapalene, Merck Frosst L 699333, Merckle ML-3000, 3M PharmaceuticalsR-840, linazolast (TMK-688), Zeneca ZD-7717, Zeneca ZM-216800, ZenecaZM-230487, Zeneca ZD-2138, and NDGA (nondihydroguaiaretic acid).

In another aspect, the invention comprises a 5-LO inhibitor and a COXinhibitor and its use. Preferably, the COX inhibitor is a selectiveCOX-1 inhibitor, i.e., that it inhibits the activity of COX-1 more thanit inhibits the activity of COX-2. The use of a 5-LO inhibitor and a COXinhibitor is intended to embrace administration of each inhibitor in asequential manner in a regimen that will provide beneficial effects ofthe drug combination, the co-administration of the inhibitors in asubstantially simultaneous manner, such as in a single capsule having afixed ratio of these active agents, or in multiple, separate capsulesfor each agent, as well as a single compound that inhibits both enzymes.

The COX inhibitor can be selected from the group consisting ofcelecoxib; rofecoxib; meloxicam; piroxicam; valdecoxib, parecoxib,etoricoxib, CS-502, JTE-522; L-745,337; FR122047; NS398; fromnon-selective NSAIDs that would include aspirin, ibuprofen, indomethacinCAY10404, diclofenac, ketoprofen, naproxen, ketorolac, phenylbutazone,tolfenamic acid, sulindac, and others, or from steroids orcorticosteroids. Compounds which selectively inhibit cyclooxygenase-2have been described in U.S. Pat. Nos. 5,380,738, 5,344,991, 5,393,790,5,466,823, 5,434,178, 5,474,995, 5,510,368 and WO documents WO96/06840,WO96/03388, WO96/03387, WO95/15316, WO94/15932, WO94/27980, WO95/00501,WO94/13635, WO94/20480, and WO94/26731, and are otherwise known to thoseof skill in the art.

Selective COX-1 inhibitors are known in the art. The following is a listof preferred COX-1 selective NSAIDs: SC-560 [Smith et al., Proceedingsof the National Academy of Sciences of the United States of America95:13313-8 (1998)], FR122047 [Dohi et al., European Journal ofPharmacology 243:179-84 (1993)], Valeroyl salicylate, Aspirin. Aspirinis an irreversible cyclooxygenase inhibitor that is rapidly inactivatedin vivo. While aspirin can inhibit COX-1 and COX-2, prior treatment withaspirin can inactivate all pre-existing COX-1 before or duringexpression of COX-2. Thus any new COX-2 that is expressed is active butall “older” COX-1 or COX-2 is inactivated.

The following is a list of NSAIDs that preferentially inhibit COX-1versus COX-2: Dexketoprofene, Keterolac, Flurbiprofen, Suprofen. Seealso [Warner et al., Proceedings of the National Academy of Sciences ofthe United States of America 96:7563-8 (1999)].

In another embodiment, the invention comprises a 5-LO inhibitor and aCOX-2 activator and its use. COX-2 activators also are known in the art.See [Tanabe and Tohnai, Prostaglandins & other Lipid Mediators68-69:95-114 (20020] for review article concerning regulation of COX-2gene expression and as a reference for those compounds or treatmentslisted below without a reference. Preferred COX-2 activators includeultrasound therapy [Sena et al., Ultrasound in Medicine & Biology31:703-8 (2005)], pulsed electromagnetic fields (PEMF) [Lohmann et al.,Journal of Orthopaedic Research 21:326-34 (2003)], BMP2 [Chikazu et al.,Journal of Bone and Mineral Research 17:1430-40 (2002)], PDGF, FGF, andPTH and its analogs (PTHrP and teraparatide) [Maciel et al., Journal ofRheumatology 24:2429-35 (1997)]. Other COX-2 activators includeProstaglandins and prostaglandin receptor agonists [Rosch et al.,Biochemical and Biophysical Research Communications 338:1171-8 (20050],PDGF (platelet derived growth factor), IL-1alpha (interleukin 1 alpha),IL-1beta, TNF-alpha (tumor necrosis factor alpha), FGF (fibroblastgrowth factor), TGF-beta (transforming growth factor beta), TGF-alpha,EGF (epidermal growth factor), TPA (tetradecanoyl phorbol acetate),

In addition, the invention comprises a combination comprising atherapeutically-effective amount of a 5-lipoxygenase inhibitor and acyclooxygenase-2 inhibitor, such as, e.g., licofelone, Dupont Dup 697,Taisho NS-398, meloxicam, flosulide, Glaxo SmithKline 406381, GlaxoSmithKline 644784, or tepoxalin.

The modulation of bone metabolism by the methods of the invention can bedetermined by examination of bone strength and mass after administrationcompared to a control subject. Such examination can be performed in situby using imaging techniques (e.g., X-ray, nuclear magnetic resonanceimaging, X-ray tomography, ultrasound, and sound conduction) or stresstesting, or ex vivo by standard histological, radiographic, mechanical,or biochemical methods. Modulation of bone density and/or bone mass canbe assessed by changes in one or more parameters such as bone mineraldensity, bone strength, trabecular number, bone size, and bone tissueconnectivity. Several methods for determining bone mineral density (BMD)are known in the art. For example, BMD measurements may be done using,e.g., dual energy xray absorptiometry or quantitative computedtomography, and the like. Similarly, increased bone formation can bedetermined using methods well known in the art. For example, dynamicmeasurements of bone formation rate (BFR) can be performed ontetracycline labeled cancellous bone from the lumbar spine and distalfemur metaphysis using quantitative digitized morphometry (Ling et al.,Endocrinology 140: 5780-5788 (1999)). Alternatively, bone formationmarkers, such as alkaline phosphatase activity, serum collagen peptidelevels, or serum osteocalcin levels can be assessed to indirectlydetermine whether increased bone formation has occurred (Looker et al.,Osteoporosis International 11: 467-480 (2000)). Compounds that modulatean arachidonic acid metabolic or signaling pathway can be tested fortheir ability to accelerate or enhance fracture healing and/or boneformation, promote bone formation, and prevent bone loss. This can betested in a variety of animal models well known to one skilled in theart such as animal fracture models, animal osteotomy models, animalskull trephine defect models, animal bone defect models, various animalssegmental defect models and bone lengthening models, ovariectomy inducedbone loss models, and the like. The utility of these animal models iswell established and is supported by a wide range of differentobservations. For example, BMP2 studies in animals including ratsdemonstrated that BMP2 stimulates osteogenesis and BMP2 is now usedclinically in humans for bone repair applications (tradename INFUSE).There are hundreds of papers about this in animals and tens of papersabout humans; NSAIDs inhibit fracture repair in rats [Simon et al.,Cyclooxygenase 2 function is essential for bone fracture healing.Journal of Bone and Mineral Research 17:963-76 (2002)] and NSAID use hasbeen correlated to poor fracture healing in humans [Burd et al., Journalof Bone and Joint Surgery (British) 85B:700-5 (2003)]; studies cited inRubin et al. (2001), JBJS 83(2):259-270 indicating that ultrasoundtreatment accelerates fracture repair in rats (Azuma ref.) and inhumans. FDA guidelines for osteoporosis therapies indicate thatpreclinical studies require use of 2 species and that one must be anovariectomized rat model.

Modulation of bone metabolism by the methods of the invention can bedetermined in vitro by examining the proliferation, survival, anddifferentiation of osteoblasts and/or chondrocytes following treatmentthat alters arachidonic acid metabolism as compared to mock treatedcells. Treatment of cells or organ explants such as newborn rodentcalvaria or phalanges can be with compounds that inhibit 5-lipoxygenaseactivity, alter cyclooxygenase activity, affect leukotriene orprostaglandin receptor function, and the like as set forth in thisapplication. Additional treatment methods can include use of antisensenucleic acids, interfering RNAs, other nucleic acid or proteins, and thelike. Osteoblast or chondrocyte proliferation and survival can bemeasured by a number of techniques well known to one skilled in the artssuch as cell counting, incorporation of radiolabeled thymidine orbromodeoxyuridine into replicating DNA, trypan blue exclusion, andterminal deoxynucleotidyl transferase end labeling of DNA within cellsundergoing apoptosis. Differentiation of osteoblasts and/or chondrocytescan be measured by a number of techniques well known to one skilled inthe arts and would include formation of mineralized nodules stained bythe method of von Kossa or with alizarin red to ascertain osteoblast orchondrocyte culture mineralization, alcian blue staining of chondrocytesto measure elaboration of proteoglycan matrix, gene expression analysesto measure markers of osteoblast and chondrocyte differentiation such asType I, Type II, and Type X collagen, osteocalcin, and aggrecan usingprotein or nucleic acid based assay methods, measurement of alkalinephosphatase activity, and measurement of RANKL, OPG, VEGF, bonemorphogenetic protein, and other growth factors by quantitative methodssuch as enzyme-linked immuno assays (EIA).

5-Lipoxygenase-Activating Protein (FLAP)

FLAP is an 18-kD membrane-bound polypeptide which specifically bindsarachidonic acid and activates 5-LO by acting as an arachidonic acidtransfer protein. The FLAP gene spans greater than 31 kb and consists offive small exons and four large exons (GenBank 182657, Genbank M60470for exon 1, Genbank M63259 for exon 2, Genbank M63260 for exon 3,Genbank M63261 for exon 4, and Genbank M6322 for exon 5).

The nuclear envelope is the intracellular site at which 5-LO and FLAPact to metabolize arachidonic acid, and ionophore activation ofneutrophils and monocytes results in the translocation of 5-LO from anonsedimentable location to the nuclear envelope. Inhibitors of FLAPfunction prevent translocation of 5-LO from cytosol to the membrane andinhibit 5-LO activation. Thus, FLAP inhibitors are anti-inflammatorydrug candidates.

Leukotriene synthesis is reduced by drugs that inhibit FLAP (MK866) orin mice lacking FLAP. Thus, in one aspect of the invention, FLAPinhibitors such as BAYx 1005, MK-886, and MK-0591, are used in methodsthat modulate an arachidonic acid metabolic or signaling pathway therebyaccelerating and/or enhancing fracture healing and bone formation.

Antisense Treatment

The term “antisense nucleic acid” is intended to refer to anoligonucleotide complementary to the base sequences of 5-LO orFLAP-encoding DNA and RNA or those that encode other proteins in anarachidonic acid metabolic or signaling pathway. Antisenseoligonucleotides can be modified or unmodified RNA, DNA, or mixedpolymer oligonucleotides, and, when introduced into a target cell,specifically bind to their target nucleic acid and interfere withtranscription, RNA processing, transport and/or translation. Targetingdouble-stranded (ds) DNA with oligonucleotide leads to triple-helixformation; targeting RNA will lead to double-helix formation.

Antisense constructs can be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. Antisense RNA constructs, or DNA encoding such antisense RNAs, canbe employed to inhibit gene transcription or translation or both withina host cell, either in vitro or in vivo, such as within a host animal,including a human subject. Nucleic acid sequences comprising“complementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules, whereguanine pairs with cytosine (G:C) and adenine pairs with either thymine(A:T) in the case of DNA, or adenine pairs with uracil (A:U) in the caseof RNA.

While all or part of the gene sequence may be employed in the context ofantisense construction, preferably any sequence 17 bases long can beused to specify a unique target sequence. Although shorter oligomers areeasier to make and increase in vivo accessibility, numerous otherfactors are involved in determining the specificity of hybridization.The antisense oligonucleotide is selected such that the binding affinityand sequence specificity to its complementary target is sufficient foruse as therapeutic agents. Thus, oligonucleotides of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more base pairs can be used.One can readily determine whether a given antisense nucleic acid iseffective at targeting of the corresponding host cell gene simply bytesting the constructs in vitro to determine whether the endogenousgene's function is affected or whether the expression of related geneshaving complementary sequences is affected.

Interfering RNA

Interfering RNA (RNAi) fragments, particularly double-stranded (ds)RNAi, can be used to modulate an arachidonic acid metabolism orsignaling pathway. Small interfering RNA (siRNA) are typically 19-25nucleotide-long RNA molecules that interfere with the expression ofgenes. Methods relating to the use of RNAi to silence genes in C.elegans, Drosophila, plants, and humans are known in the art (Fire etal., Nature 391: 806-811 (1998); Sharp, P. A. RNA interference 2001.Genes Dev. 15: 485-490 (2001); Tuschl, T. Chem. Biochem. 2: 239-245(2001); WO0129058; and WO9932619).

The nucleotide sequence employed RNAi comprises sequences that are atleast about 15 to 50 basepairs. The sequence can be a duplex, optionallywith overhangs at the 5′-end and/or the 3′-end, where one strand of theduplex comprises a nucleic acid sequence of at least 15 contiguous baseshaving a nucleic acid sequence of a nucleic acid molecule within anarachidonic acid metabolic or signaling pathway. The length of eachstrand can be longer where desired, such as 19, 20, 21, 22, 23, 24, 25,or 30 nucleotides or up to the full length of any of those describedherein. The single-stranded overhang can be, for example, 1, 2, 3, 4, 5,or 10 nucleotides long, and can be present at the 3′-end, the 5′ end, orboth the 3′-end and the 5′-end. Such fragments can be readily preparedby directly synthesizing the fragment by chemical synthesis, byapplication of nucleic acid amplification technology, or by introducingselected sequences into recombinant vectors for recombinant production.

In particular, the nucleotide sequences or RNAi can be oligonucleotidescomplementary to the base sequences of 5-LO or FLAP-encoding DNA and RNAor to the base sequences encoding other proteins in an arachidonic acidmetabolism or signaling pathway. The oligonucleotides can be modified orunmodified RNA, DNA, or mixed polymer oligonucleotides, and, whenintroduced into a target cell, specifically bind to their target nucleicacid and interfere with transcription, RNA processing, transport and/ortranslation.

Other Agents

In another aspect of the invention, an additional agent or drug may beadministered to the subject. The additional agent can contain one ormore active agents that effectively regulate calcium homeostatis,modulate chondrogenesis, modulate osteogenesis, modulate boneremodeling, regulate pain, regulate inflammation, or have antibioticactivity. The additional active agent can be, but is not limited to, anestrogen, an IGF, insulin, bone morphogenetic proteins and other growthfactors, osteoprotegrin (OPG), a calcitonin, a bisphosphonate, vitaminD₃ or an analogue thereof, a statin, an adrogen, a fluoride salt, aparathyroid hormone or an analogue thereof, agents that enhanceangiogenesis such as vascular endothelial growth factor (VEGF), agentsthat alter regulation of transcription of naturally occurring hormoneregulators involved in bone metabolism, a vitamin, a mineral supplement,a nutritional supplement, and combinations thereof. The additional agentalso may be an antibiotic such as gentamycin, ciprofloxacin, vancomycin,and/or others. This additional active agent can be administered to thesubject prior to, concurrently with or subsequently to administration ofthe 5-lipoxygenase inhibitor of this invention. Anti-inflammatory drugs,including but not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, p38 kinase inhibitors, and antiviral drugs, includingbut not limited to ribivirin, vidarabine, acyclovir and ganciclovir, mayalso be combined in compositions of the invention. Antibiotic compoundsincluding but not limited to gentamicin, teicoplanin, tobramycin, andvancomycin, may also be combined in the composition of the invention.

III. PHARMACEUTICAL FORMULATIONS AND MODES OF ADMINISTRATION

The methods described herein use pharmaceutical compositions comprisingthe molecules described above, together with one or morepharmaceutically acceptable excipients or vehicles, and optionally othertherapeutic and/or prophylactic ingredients. Such excipients includeliquids such as water, saline, glycerol, polyethyleneglycol, hyaluronicacid, ethanol, cyclodextrins, modified cyclodextrins (i.e., sulfobutylether cyclodextrins) etc. Suitable excipients for non-liquidformulations are also known to those of skill in the art.Pharmaceutically acceptable salts can be used in the compositions of thepresent invention and include, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. A thorough discussion of pharmaceuticallyacceptable excipients and salts is available in Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990).

Additionally, auxiliary substances, such as wetting or emulsifyingagents, biological buffering substances, surfactants, and the like, maybe present in such vehicles. A biological buffer can be virtually anysolution which is pharmacologically acceptable and which provides theformulation with the desired pH, i.e., a pH in the physiologicallyacceptable range. Examples of buffer solutions include saline, phosphatebuffered saline, Tris buffered saline, Hank's buffered saline, and thelike.

Depending on the intended mode of administration, the pharmaceuticalcompositions may be in the form of solid, semi-solid or liquid dosageforms, such as, for example, tablets, suppositories, pills, capsules,powders, liquids, suspensions, creams, ointments, lotions or the like,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include an effective amount of theselected drug in combination with a pharmaceutically acceptable carrierand, in addition, may include other pharmaceutical agents, adjuvants,diluents, buffers, etc.

The invention includes a pharmaceutical composition comprising acompound of the present invention including isomers, racemic ornon-racemic mixtures of isomers, or pharmaceutically acceptable salts orsolvates thereof together with one or more pharmaceutically acceptablecarriers, and optionally other therapeutic and/or prophylacticingredients.

In general, compounds of this invention will be administered aspharmaceutical formulations including those suitable for oral (includingbuccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal orparenteral (including intramuscular, intraarterial, intrathecal,subcutaneous and intravenous) administration, in a form suitable foradministration by inhalation or insufflation, or in a form suitable foradministration at the bone formation site. The preferred manner ofadministration is oral or intravenous using a convenient daily dosageregimen which can be adjusted according to the degree of affliction.

Formulations for delivery at the bone formation site include adsorptiononto or encapsulation within polylactide and/or polygalactide polymers,palmitic acid, alginate, plaster, calcium sulfate, calcium phosphate,mixtures of calcium sulfate and calcium phosphate, hydroxyapatite,collagen or other extracellular matrix material, bone wax (such as thatfrom CP Medical, Inc., Ethicon, Inc., Unites States Surgical Corp., orCeremed), Orthocon Bone Putty (a mixture of calcium stearate, vitamin Eacetate, and alkylene oxide copolymer) or other materials or compoundsthat can be used for this purpose. Delivery can be accomplished bydirect placement at the bone formation site or by deposition of theactive compound of the invention with or without a carrier onto thesurface of prosthetic or surgically implanted devices.

A pharmaceutically or therapeutically effective amount of thecomposition is delivered to the subject. The precise effective amountvaries from subject to subject and depends upon the species, age, thesubject's size and health, the nature and extent of the condition beingtreated, recommendations of the treating physician, and the therapeuticsor combination of therapeutics selected for administration. Thus, theeffective amount for a given situation can be determined by routineexperimentation. For purposes of the present invention, generally atherapeutic amount will be in the range of about 0.05 mg/kg to about 40mg/kg body weight, more preferably about 0.5 mg/kg to about 20 mg/kg, inat least one dose. In larger mammals the indicated daily dosage can befrom about 1 mg to 4,800 mg, one or more times per day, more preferablyin the range of about 10 mg to 1,200 mg. The subject may be administeredas many doses as is required to reduce and/or alleviate the signs,symptoms, or causes of the disorder in question, or bring about anyother desired alteration of a biological system. One of ordinary skillin the art of treating such diseases will be able, without undueexperimentation and in reliance upon personal knowledge and thedisclosure of this application, to ascertain a therapeutically effectiveamount of the compounds of this invention for a given disease. Whenpracticing the methods of the invention starting human doses may need tobe estimated from rat dose data. Such estimation methods are well knownin the art. See FDA publication “Guidance for Industry: Estimating theMaximum Safe Starting Dose in Initial Clinical Trials for Therapeuticsin Adult Healthy Volunteers” published July 2005 (Federal RegisterDocument 5-14456) and available online atwww.fda.gov/Cder/guidance/5541fnl.pdf. In general, the rat doseexpressed as mg/kg should be divided by 6.2 to obtain an equivalenthuman dose.

When desired, formulations can be prepared with enteric coatings adaptedfor sustained or controlled release administration of the activeingredient.

The pharmaceutical preparations are preferably in unit dosage forms. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

IV. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 5-LO Knock Out Mice

Knock out mice lacking 5-lipoxygenase (Alox5−/− or 5-LO−/−) werepurchased from Jackson Laboratory, Bar Harbor, Me. An impending femurfracture was stabilized with an intramedullary wire that was insertedretrograde into the femoral canal. A three-point bending device was usedto make the fracture. Femur fracture healing was measured or assessed byhistomorphometry, radiography, and torsional mechanic testing. The5-LO−/− mice demonstrated statistically significant, quantitativeacceleration and enhancement of fracture healing as compared towild-type mice of identical genetic background and age (C57BL/6). Closedmid-diaphyseal fractures were made in 10-12 week old female mice.Fracture healing was assessed by x-rays (FIG. 3) and quantitativelyassessed by torsional mechanical testing 4 and 12 weeks after fracture(FIG. 4 and TABLE 2). After 4 or 12 weeks of healing, the fracturedfemurs from 5LO−/− and wild type (WT) mice were excised and mechanicallytested to failure in torsion using an MTS servohydraulic test machineand Interface 20 Nm torque load cell. Fractured femur dimensions weremeasured before and after testing. Peak torque, rigidity, maximum shearstress, and shear modulus were calculated from callus dimensions and thetorque to angular displacement curves. All mechanical parameters were50-120% higher after 4 weeks of healing in the 5-LO−/− as compared tothe WT mice. Histomorphometric analysis of time-staged fracturespecimens from normal and 5-LO−/− mice showed that cartilage area peakedearly and to a greater extent in the 5-LO−/− mice (FIG. 5 and TABLE 3).Further, significantly more new bone (mineralized tissue) was present inthe 5-LO−/− fracture callus at 7 and 10 days after fracture. The datademonstrate that fracture healing is accelerated and enhanced in the5LO-KO mice.

TABLE 2 Summary of fractured femur torsional mechanical testing datafrom 5-LO−/− and wild-type mice of identical genetic background and ageat time of fracture (Fx). Mean Percentages (Fracture/Contralateral) ± SDSample Days Peak Max. Shear Shear Strain Size Post-Fx Torque RigidityStress Modulus C57BL/6 9 28  85.3 ± 16.7  61.9 ± 31.3 18.2 ± 5.4  7.8 ±4.4 C57BL/6 6 84  77.8 ± 20.1 110.6 ± 19.1 26.2 ± 12.8 25.9 ± 13.4Lox5−/− 8 28 128.5 ± 30.3 109.9 ± 37.4 31.8 ± 9.0  17.4 ± 11.1 Lox5−/− 884 131.4 ± 26.0  95.8 ± 37.8 49.8 ± 16.9 23.2 ± 13.2

TABLE 3 Summary of fracture callus histomorphometric analysis from5-LO−/− and wild-type mice of identical genetic background and age attime of fracture. Percent Cartilage Percent Mineralized Tissue (mean ±S.D.) (mean ± S.D.) Time Point Wild-Type 5-LO−/− P value Wild-Type5-LO−/− P value  7 days 7.84 ± 1.31 30.84 ± 3.46 <0.001 12.89 ± 3.7624.56 ± 3.33 <0.001 10 days 20.16 ± 6.13  14.46 ± 7.53 0.226 35.49 ±9.67 47.57 ± 2.86 0.028 14 days 3.63 ± 1.37  2.73 ± 3.71 0.625 44.66 ±7.14 51.46 ± 5.38 0.127 21 days 0 ± 0  0 ± 0 1.000 77.26 ± 6.26 75.72 ±2.55 0.624

The serial x-rays (FIG. 3) show that fracture healing is accelerated inthe 5LO−/− mice as compared to wild type mice (C57BL/6). Morespecifically, the 10 day old fracture from the 5LO−/− mouse appears tobe at similar stage as the 14 day old fracture from the wild type mouse,the 14 day 5LO−/− fracture is similar to the 21 day wild type fracture,and the 1 month 5LO−/− fracture is similar to a 3 month old wild typefracture. The mechanical testing data show quantitatively that thestructural and material properties of the 5-LO−/− fracture callus werestatistically significantly better than the controls after 4 weeks ofhealing with a 50% increase in peak torque, a 75% increase in rigidity,a 75% increase in maximum shear stress, and over a 100% increase inshear modulus. Further, the 4 week mechanical testing parameters fromthe 5LO−/− mice were similar to those from the 12 week wild type mice,supporting the x-ray data of FIG. 3 and demonstrating that fracturehealing was accelerated and enhanced in the 5LO−/− mice. After 12 weekof healing, the rigidity and shear modulus of the wild-type fracturecallus had caught-up with the 5-LO−/− fracture callus. Histomorphometricmeasurements of time-staged fracture callus specimens from the 5-LO−/−and WT mice support the mechanical and radiographic observations (FIG. 5and TABLE 2). Callus cartilage area peaked by day 7 post-fracture in the5-LO−/− mice but not until day 10 in the WT mice. There was almost4-times more cartilage present in the 5-LO−/− callus at day 7 ascompared to that from the WT mice. Concurrently, more new bone formationalso occurred in the 5-LO−/− mice with almost twice as much new bone(mineralized tissue) present at day 7 and 30% more new bone at day 10 ascompared to the WT mice. The data is thus consistent with fracturehealing occurring faster and producing more mechanically sound fracturecallus with enhanced structural and material properties in the 5-LO−/−mice than in normal mice.

Example 2 COX-2 Knockout Mice

Fracture healing was assayed in mice with a targeted deletion of theCOX-2 gene. Closed, mid-diaphyseal femur fractures were made in theright hindlimb of COX-2 knockout, COX-1 knockout, and wild type mice(not shown). Fracture healing was assessed by x-rays and histology (FIG.6), and by mechanical testing (not shown). The data show that fracturehealing was dramatically impaired in the COX-2 knockout mice, but notthe COX-1 knockout or wild type mice. X-rays after 14 days of healingshow a large mineralized fracture callus in the COX-1 knockout mouse(FIG. 6) with little or no evident mineralized callus in the COX-2knockout mouse. Histological examination confirmed the x-ray findings inthat the COX-2 knockout callus had a significant amount of cartilage butno new bone was evident. Torsional mechanical testing data shows thatfracture callus structural and material properties are statisticallysignificantly worse than COX-1 knockout or wild type mice. When combinedwith the experimental results of example 1, example 3, and example 4this demonstrates how arachidonic acid metabolic or signaling can bemanipulated according to the methods of the invention to affect boneformation.

Example 3 Treatment of Rats with a 5-Lipoxygenase Inhibitor

Sprague-Dawley rats (3 months old) underwent a standard closed femurfracture procedure as described in the art (Simon et al. Journal of Boneand Mineral Research, 17(6): 963-976 (2002); Bonnarens and Einhorn,Production of a standard closed fracture in laboratory animal bone.Journal of Orthopaedic Research, 2: 97-101 (1984)). The impendingfracture was stabilized with an intramedullary stainless steel pin.Beginning 4 hours after fracture the rats were treated with 30 mg/kg ofNDGA (nordihydrogaiaretic acid) in 1% methylcellulose (5-lipoxygenaseinhibitor treatment group) or with carrier only (1% methylcellulose).The day after surgery and continuing until day 14 post-fracture,experimental rats were treated with 2 doses of NDGA (30 mg/kg), thefirst dose between 8-10 AM and then again with another NDGA dose 8-10hours later. Control rats were treated similarly but with carrier only(1% methylcellulose). Three weeks after fracture, the rats weresacrificed, the fractured femurs were harvested, and high resolutionradiographs were made of the fractured femurs using a Packard Faxitronand Kodak MinR2000 mammography film. Two representative radiographs areshown in FIG. 7 for each treatment group: control and 5-lipoxygenase(5-LO) inhibitor treated.

The radiographs show that after 3 weeks the fractured femurs of the 5-LOinhibitor treated rats were bridged with new bone. In contrast, awell-formed, mineralized fracture callus has formed in the control ratsbut the fracture site had not yet bridged with new bone. In rat C, thefracture is bridged with new bone on the medial (top) and lateral(bottom) sides of the fracture callus. In rat D, the fracture is bridgedwith new bone on the lateral side (bottom) and shows indications of newbone bridging on the medial side. No new bone bridging is evident in thecontrol rats (rats A and B). The data thus demonstrates that 5-LOinhibitor therapy can accelerate the fracture healing process in young,normal rats.

Example 4 Treatment of Rats with 5-Lipoxygenase Inhibitors

Sprague-Dawley rats (3 months old) underwent a standard closed femurfracture procedure as described in the art (Simon et al. Journal of Boneand Mineral Research, 17(6): 963-976 (2002); Bonnarens and Einhorn,Production of a standard closed fracture in laboratory animal bone.Journal of Orthopaedic Research, 2: 97-101 (1984)). The impendingfracture was stabilized with an intramedullary stainless steel pin.Beginning 4 hours after fracture the rats were treated with vehicle (1%methylcellulose) or inhibitors of 5-LO suspended in 1% methylcelluloseInhibitor A (NDGA) was administered at 30 mg/kg and Inhibitor B (AA-861)was administered at 5 mg/kg. The day after surgery and continuing untilday 21 post-fracture, experimental rats were treated with 2 doses ofinhibitor (either A or B), the first dose between 8-LOAM and then againwith another dose 8-10 hours later. Control rats were treated similarlybut with carrier only (1% methylcellulose). Three weeks after fracturethe rats were anesthetized and high resolution radiographs were made ofthe fractured femurs using a Packard Faxitron and Kodak MinR2000mammography film (FIGS. 8A, 8B, and 8C). Five weeks after fracture therats were sacrificed, femurs resected, and assayed for structuralmechanical properties by torsional mechanical testing (FIG. 8D).

The radiographs showed that after 3 weeks of healing, the fracturesappeared bridged in the 5-LO inhibitor treated rats but not in thevehicle treated rat.

Torsional mechanical testing was used to measure the peak torquesustained by each femur after 5 weeks of healing. The data show that thefemurs from the Inhibitor A (NDGA) treated rats and from the Inhibitor Btreated rats had 22% and 53% greater peak torque than vehicle treatedrats (FIG. 8D). In addition, all of the femurs from the Inhibitor A or Btreated rats failed as boney unions while 13% (2 of 15) of the femursfrom the vehicle treated rats failed as non-unions with no apparent bonebridging.

These experimental observations demonstrate that 5-LO inhibition therapycan accelerate (faster bone bridging) and enhance (better mechanicalproperties) fracture healing.

Example 5 Ex Vivo Treatment Methods Using Small Molecule Compounds,RNAi, and Antisense Compounds

Methods to promote ex vivo osteogenesis are used, e.g., to aid inhealing of recalcitrant bone fractures, segmental defects caused bytraumatic injuries or pathological resection of bone segments, or forjoint arthrodesis. In these instances, precursor bone cells are isolatedfrom a subject or from a suitable donor and are cultured ex vivo usingstandard methods. The cells are grown in or seeded into an appropriatescaffold that either represents the segment of missing bone or can bemolded to fit the missing segment or juxtapose the ends of the bone. Thecells are induced to form bone ex vivo using appropriate cell cultureconditions or with inductive factors, such as bone morphogeneticprotein-2 (BMP-2). Once the cells have begun to elaborate a new bonematrix, the construct can be implanted into the patient to effectosteogenesis and promote healing. This sequence of events is typicallyreferred to as a tissue engineering approach to enhancing osteogenesis.

Inhibition of 5-lipoxygenase (5-LO) can be used to promote ex vivo boneformation for tissue engineering application. This is accomplished bypromoting osteogenesis ex vivo with small molecule inhibitors of 5-LO orFLAP alone or in combination with well known inductive agents, such asBMP-2.

A second approach uses RNAi technology to inhibit 5-LO activity andpromote ex vivo osteogenesis. This is accomplished by transfecting thecultured precursor skeletal cells with pools of siRNA sequences usingcommercially available transfection reagents, such as TransIT-TKO orjetSI. Approximately 1 million cells are transfected with a cocktail of3 siRNAs specific for 5-LO or FLAP using 50-200 pmoles of each siRNA.Alternatively, a pool of siRNAs that target 5-LO and FLAP is used. As acontrol, cells are transfected similarly with commercially availablesiRNAs developed to knock-down enhanced green fluorescent protein(EGFP). Knock-down of 5-LO or FLAP is confirmed by western blot analysisand the results quantified to insure a greater than 80% reduction in5-LO and/or FLAP expression.

The treated precursor skeletal cells are cultured and osteogenesis isassessed as extracellular matrix production of cartilage or bone matrixusing measures such as alcian blue or alizarin red binding asappropriate or measures of specific matrix protein. Knock-down of 5-LOor FLAP promotes osteogenesis based upon enhanced calcified matrixdeposition measured by alizarin red binding. This indicates that an RNAior anti-sense approach to inhibiting 5-LO activity is useful forpromoting osteogenesis ex vivo for purposes of tissue engineering.

Pools of siRNA pairs for 5-LO can be chosen, e.g., from POOL-A (5′-AACTGG GCG AGA TCC AGC TGG-3′ (SEQ ID NO: 9), 5′-AAG CTC CCG GTG ACC ACGGAG-3′ (SEQ ID NO: 10), 5′-AAG GAA GCC ATG GCC CGA TTC-3′) (SEQ ID NO:11), POOL-B (5′-AAT CGA GAA GCG CAA GTA CTG-3′ (SEQ ID NO: 12), 5′-AAGGAG TGG ACT TTG TTC TGA-3′ (SEQ ID NO: 13), 5′-AAC TTC GGC CAG TAC GACTGG-3′) (SEQ ID NO: 14), or POOL-C (5′-AAG TTG GCC CGA GAT GAC CAA-3′(SEQ ID NO: 15), 5′-AAC ACA TCT GGT GTC TGA GGT-3′ (SEQ ID NO: 16),5′-AAC CAT GCG AGC CCC GCC ACC-3′) (SEQ ID NO: 17). Pools of siRNA pairsfor FLAP can be chosen, e.g., from POOL-D (5′-AAG CAA ACA TGG ATC AAGAAA-3′ (SEQ ID NO: 18), 5′-AAG TTC CTG CTG CGT TTG CTG-3′ (SEQ ID NO:19), 5′-AAT TCA GCT CTT GAG AGC ATT-3′) (SEQ ID NO: 20), POOL-E (5′-AATGGA TTC TTT GCC CAT AAA-3′ (SEQ ID NO: 21), 5′-AAG TAC TTT GTC GGT TACCTA-3′ (SEQ ID NO: 22), 5′-AAT CTA TTG GCC ATC TGG GCT-3′) (SEQ ID NO:23), or POOL-F (5′-AAC CAG AAC TGT GTA GAT GCG-3′ (SEQ ID NO: 24),5′-AAG TGA CTT TGA AAA CTA CAT-3′ (SEQ ID NO: 25), 5′-AAT GAT GTC ATGTCA GCT CCG-3′) (SEQ ID NO: 26). For brevity, only the sense strand ofeach siRNA pair is shown. It is well known in the art that siRNA pairsare double stranded small RNAs that have a 5′-AA overhang on the sensestrand and a 5′-UU overhang on the antisense strand. It also is wellknown in the art that backbone chemistry modifications can beadvantageous for stabilizing or improving the uptake of the siRNAmolecules. Pirollo K F et al., (2003), Rait A, Sleer L S, Chang E H,“Antisense therapeutics: from theory to clinical practice,” PharmacolTher. 99(1):55-77. Manufacture of oligonucleotides with advantageousbackbone chemistry modifications is within the level of ordinary skill,and use of such modified—backbone compounds (as well asnon-modified-backbone compounds) is within the scope of the presentinvention.

One skilled in the art will recognize that in addition to directtransfection of the siRNAs into cells, expression vectors can bedeveloped that express these or similar sequences and the expressionvectors delivered to the cells by transfection, viral mediated delivery,or methods for delivering DNA molecules into cells. The expressionvectors express the siRNAs leading to sustained inhibition of 5-LO,FLAP, or both and thereby promoting osteogenesis.

One skilled in the art also will recognize that additional strategies toinhibit expression of 5-LO or FLAP can be used to promote the sameosteogenic effects in the precursor skeletal cells. Such technologiesinclude use of anti-sense.

Exemplary 5-Lipoxygenase anti-sense sequences include, e.g., 5′-GCA GGTGCT TCT CGC TGC AGC C-3′ (SEQ ID NO: 27), 5′-GCC AGT ACT TGC GCT TCTCG-3′ (SEQ ID NO: 28), 5′-CCA TCG ATA TTG TTT TTG CC-3′ (SEQ ID NO: 29),5′-GGA GCT TCT CGG GCA GCT CTG TGC-3′ (SEQ ID NO: 30), 5′-CCA GGT TCTTAT ACA GCA AGC-3′ (SEQ ID NO: 31), 5′-CCA GCA GCT TGA AAA TGG GGT GC-3′(SEQ ID NO: 32), 5′-GCC CCG GGC CTT GAT GGC C-3′ (SEQ ID NO: 33), 5′-CCACGC CCT TGG CAG TCG G-3′ (SEQ ID NO: 34), and 5′-GCG GAA TCG GGC CAT GGCTTC C-3′ (SEQ ID NO: 35).

Exemplary FLAP anti-sense sequences include, e.g., 5′-GTT CCG GTC CTCTGG AAG CTC C-3′ (SEQ ID NO: 36), 5′-CGC AGA CCA GAG CAC AGC G-3′ (SEQID NO: 37), 5′-GCA AAC GCA GCA GGA AC-3′ (SEQ ID NO: 38), 5′-CGT TTC CCAAAT ATG TAG CC-3′ (SEQ ID NO: 39), 5′-GTT TTC AAA GTC ACT TCC G-3′ (SEQID NO: 40), 5′-GGT TAA CTC AAG CTG TGA AGC-3′ (SEQ ID NO: 41), 5′-GGAGCT GAC ATG ACA TC-3′ (SEQ ID NO: 42), and 5′-GGC CAC GGT CAT GTT CAAGG-3′ (SEQ ID NO: 43).

Thus, novel methods for promoting osteogenesis to accelerate or enhancebone fracture healing, treat bone defects, and enhance bone formationare disclosed. Although preferred embodiments of the subject inventionhave been described in some detail, it is understood that obviousvariations can be made without departing from the spirit and the scopeof the invention as defined by the appended claims.

I claim:
 1. A method for treating a bone fracture or a bone defect in amammalian subject, comprising: administering to said subject apharmaceutically effective amount of an antisense compound, wherein saidantisense compound reduces the activity of cyclooxygenase-1 (COX-1). 2.The method of claim 1, wherein the condition is a bone fracture.
 3. Themethod of claim 1, wherein the condition is a bone fracture selectedfrom the group consisting of a non-osteoporotic fracture, anosteoporotic fracture, a fracture associated with a congenital disease,a fracture associated with an acquired disease, or an osteotomicfracture.
 4. The method of claim 3, wherein the bone fracture is anon-osteoporotic fracture.
 5. The method of claim 3, wherein the bonefracture is an osteoporotic fracture.
 6. The method of claim 3, whereinthe bone fracture is an osteotomic fracture.
 7. The method of claim 1,wherein the condition is a bone defect.
 8. The method of claim 1,wherein administration is in vivo.
 9. The method of claim 1, whereinadministration is ex vivo.
 10. The method of claim 1, further comprisingadministering a pharmaceutically effective amount of a second compoundthat reduces a 5-lipoxygenase activity.
 11. The method of claim 10,wherein the second compound reduces a 5-lipoxygenase activity byinhibiting a five lipoxygenase activating protein (FLAP).
 12. The methodof claim 11, wherein the second compound that reduces a 5-lipoxygenaseactivity comprises a small molecule.
 13. The method of claim 12, whereinthe small molecule is[(R)(+)N′-[[5-(4-fluorophenoxy)furan-2-yl]-1-methyl-2-propynyl]-N-hydroxyurea.14. The method of claim 12, wherein the small molecule is Abbott® ABT761.
 15. The method of claim 12, wherein the small molecule is selectedfrom the group consisting of3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoicacid;3-(1-(4-chlorobenzyl)-3-(1-butyl-thio)-5-(quinolin-2-yl-methoxy)-indol-2-yl)-2,2-dimethylpropanoic acid); nordihydroguaiaretic acid;2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone;(N-(1-benzo(b)thien-2-ylethyl)-N-hydroxyurea); masoprocol; tenidap;flobufen; lonapalene; tagorizine; Abbott® A-121798; Abbott® A-76745;Abbott® A-78773;[(R)(+)N′-[[5-(4-fluorophenoxy)furan-2-yl]-1-methyl-2-propynyl]-N-hydroxyurea;Abbott® ABT 761; Dainippon® AL-3264; Bayer® Bay-x-1005; Biofor® BF-389;bunaprolast; Cytomed® CMI-392; Takeda® CV-6504; enazadrem phosphate; LeoDenmark® ETH-615; flezelastine hydrochloride; Merck Frosst® L-663536;Merckle® ML-3000; 3M Pharmaceuticals® R-840; rilopirox; Schering Plough®SCH-40120; tepoxalin; linazolast; Zeneca® ZD-2138; Bristol-Myers Squibb®BU-4601A; carbazomycin C; lagunamycin; Wellcome® BW-70C; Ciba-Geigy®CGS-26529; Warner-Lambert® CI 1004; Warner-Lambert® PD-136005;Warner-Lambert® PD-145246; Elsai® E-3040; Fujirebio® F-1322; Fujisawa®FR-110302; Merck Frosst® L-699333; Merck Frosst® L-739010; Lilly®LY-269415; Lilly® LY-178002; Hoechst Roussel® P-8892; SmithKlineBeecham® SB-202235; American Home Products® WAY-121520; American HomeProducts® WAY-125007; Zeneca® ZD-7717; Zeneca® ZM-216800; Zeneca®ZM-230487;1,2-dihydro-n-(2-thiazolyl)-1-oxopyrrolo(3,2,1-kl)phenothiazine-1-carboxamide;Abbott® A-65260; Abbott® A-69412; Abbott® A-63162; American HomeProducts® AHR-5333; Bayer® Bay-q-1531; Boehringer Ingelheim® BI-L-357;Boehringer Ingelheim® BI-L-93BS; Boehringer Ingelheim® BIL 226XX;Bristol-Myers Squibb® BMY-30094; carbazomycin B; Wellcome® BW-B218C;Chauvin® CBS-1114; Ciba-Geigy® CGS-21595; Ciba-Geigy® CGS-22745;Ciba-Geigy® CGS-23885; Ciba-Geigy® CGS 24891; Ciba-Geigy® CGS-8515;Chiesi® CHF-1909; Warner-Lambert® CI-986; Warner-Lambert® CI-987;cirsiliol; docebenone; Eisai® E-5110; Eisai® E-6080; enofelast;epocarbazolin-A; eprovafen; evandamine; Fisons® FPL 62064; Zeneca®ICI-211965; Zeneca® ICI-216800; Kyowa Hakko® KF-8940; Merck® L-651392;Merck® L-651896; Merck® L-652343; Merck® L-656224; Merck® L-670630;Merck® L-674636; Lilly® LY-233569; Merck® MK-591; Merck® L-655240;nitrosoxacin-A; Ono® ONO-5349; Ono® ONO-LP-219; Ono® ONO-LP-269;Warner-Lambert® PD-127443; Purdue Frederick® PF-5901; Rhone-PoulencRorer® Rev-5367; Rhone-Poulenc Rorer® RG-5901-A; Rhone-Poulenc Rorer®RG-6866; Roussel-Uclaf® RU-46057; Searle® SC-41661A; Searle® SC-45662;Sandoz® SDZ-210-610; SmithKline Beecham® SK&F-104351; SmithKlineBeecham® SK&F-104493; SmithKline Beecham® SK&F-105809; Synthelabo®SL-81-0433; Teijin® TEI-8005; Terumo® TMK-777; Terumo® TMK-781; Terumo®TMK-789; Terumo® TMK-919; Terumo® TMK-992; Teikoku Hormone® TZI-41127;American Home Products® WAY-120739; American Home Products® WY-47288;American Home Products® WY-48252; American Home Products® WY-50295;Yoshitomi® Y-19432;4-{3-[4-(2-methyl-1H-imidazol-1-yl)phenylthio]}phenyl-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide;esculetin; phenidone; Boehringer Ingelheim® BI-L-239;5,8,11-eicosatriynoic acid; 5,8,11,14-eicosatetraynoic acid;cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate; curcumin; gossypol; caffeicacid; baicalein; 7,7-dimethyleicosadrenoic acid; Lilly® LY-311727;bromoenol lactone; methyl arachidonyl fluorophosphonate; methyly-linolenyl fluorophosphonate; oleyoxyethyl phosphorylcholine;arachidonyl trifluoromethyl ketone; n-(p-amylcinnamoyl) anthranilicacid; mepacrine; quinacrine; atabrine; parabromophenacylbromide;aristolochic acid; cortisone; Glaxo SmithKline® SB-480848; GlaxoSmithKline® SB-659032; Glaxo SmithKline® SB-677116; Bristol-MyersSquibb® BMS-181162; Sterling-Winthrop® MJ33; and MillenniumPharmaceuticals® MLN977.
 16. The method of claim 12, wherein the smallmolecule is selected from the group consisting of masoprocol; tenidap;(N-(1-benzo(b)thien-2-ylethyl)-N-hydroxyurea); flobufen; lonapalene;tagorizine;2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone;Abbott® A-121798; Abbott® A-76745; Abbott® A-78773;[(R)(+)N′-[[5-(4-fluorophenoxy)furan-2-yl]-1-methyl-2-propynyl]-N-hydroxyurea;Abbott® ABT 761; Dainippon® AL-3264; Bayer® Bay-x-1005; Biofor® BF-389;bunaprolast; Cytomed® CMI-392; Takeda® CV-6504; Ciba-Geigy® CGS-26529;enazadrem phosphate; Leo Denmark® ETH-615; flezelastine hydrochloride;Merck Frosst® L-663536; Merck Frosst® L-699333; Merckle® ML-3000, 3MPharmaceuticals® R-840; rilopirox; Schering Plough® SCH-40120;tepoxalin; linazolast; Zeneca® ZD-7717; Zeneca® ZM-216800; Zeneca®ZM-230487; Zeneca® ZD-2138; and nordihydroguaiaretic acid.
 17. Themethod of claim 12, wherein the small molecule is selected from thegroup consisting of tenidap;(N-(1-benzo(b)thien-2-ylethyl)-N-hydroxyurea); flobufen; lonapalene;tagorizine;2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone;Abbott® A-121798; Abbott® A-76745; Abbott® A-78773;[(R)(+)N′-[[5-(4-fluorophenoxy)furan-2-yl]-1-methyl-2-propynyl]-N-hydroxyurea;Abbott® ABT 761; Ciba-Geigy® CGS-26529; Biofor® BF-389; Cytomed®CMI-392; Leo Denmark® ETH-615; Merck Frosst® L 699333; Merckle® ML-3000;3M Pharmaceuticals® R-840; linazolast; Zeneca® ZD-7717; Zeneca®ZM-216800; Zeneca® ZM-230487; Zeneca® ZD-2138, and nordihydroguaiareticacid.
 18. The method of claim 11, wherein the second compound thatreduces a 5-lipoxygenase activity comprises a nucleic acid comprising asequence selected from the group consisting of 5′-AAC TGG GCG AGA TCCAGC TGG-3′ (SEQ ID NO: 9), 5′-AAG CTC CCG GTG ACC ACG GAG-3′ (SEQ ID NO:10), 5′-AAG GAA GCC ATG GCC CGA TTC-3′ (SEQ ID NO: 11), 5′-AAT CGA GAAGCG CAA GTA CTG-3′ (SEQ ID NO: 12), 5′-AAG GAG TGG ACT TTG TTC TGA-3′(SEQ ID NO: 13), 5′-AAC TTC GGC CAG TAC GAC TGG-3′ (SEQ ID NO: 14),5′-AAG TTG GCC CGA GAT GAC CAA-3′ (SEQ ID NO: 15), 5′-AAC ACA TCT GGTGTC TGA GGT-3′ (SEQ ID NO: 16), 5′-AAC CAT GCG AGC CCC GCC ACC-3′ (SEQID NO: 17), 5′-AAG CAA ACA TGG ATC AAG AAA-3′ (SEQ ID NO: 18), 5′-AAGTTC CTG CTG CGT TTG CTG-3′ (SEQ ID NO: 19), 5′-AAT TCA GCT CTT GAG AGCATT-3′ (SEQ ID NO: 20), 5′-AAT GGA TTC TTT GCC CAT AAA-3′ (SEQ ID NO:21), 5′-AAG TAC TTT GTC GGT TAC CTA-3′ (SEQ ID NO: 22), 5′-AAT CTA TTGGCC ATC TGG GCT-3′ (SEQ ID NO: 23), 5′-AAC CAG AAC TGT GTA GAT GCG-3′(SEQ ID NO: 24) 5′-AAG TGA CTT TGA AAA CTA CAT-3′ (SEQ ID NO: 25), and5′-AAT GAT GTC ATG TCA GCT CCG-3′ (SEQ ID NO: 26).
 19. The method ofclaim 10, wherein the second compound that reduces a 5-lipoxygenaseactivity comprises a nucleic acid comprising a sequence selected fromthe group consisting of 5′-GCA GGT GCT TCT CGC TGC AGC C-3′ (SEQ ID NO:27), 5′-GCC AGT ACT TGC GCT TCT CG-3′ (SEQ ID NO: 28), 5′-CCA TCG ATATTG TTT TTG CC-3′ (SEQ ID NO: 29), 5′-GGA GCT TCT CGG GCA GCT CTG TGC-3′(SEQ ID NO: 30), 5′-CCA GGT TCT TAT ACA GCA AGC-3′ (SEQ ID NO: 31),5′-CCA GCA GCT TGA AAA TGG GGT GC-3′ (SEQ ID NO: 32), 5′-GCC CCG GGC CTTGAT GGC C-3′ (SEQ ID NO: 33), 5′-CCA CGC CCT TGG CAG TCG G-3′ (SEQ IDNO: 34), 5′-GCG GAA TCG GGC CAT GGC TTC C-3′ (SEQ ID NO: 35), 5′-GTT CCGGTC CTC TGG AAG CTC C-3′ (SEQ ID NO: 36), 5′-CGC AGA CCA GAG CAC AGCG-3′ (SEQ ID NO: 37), 5′-GCA AAC GCA GCA GGA AC-3′ (SEQ ID NO: 38),5′-CGT TTC CCA AAT ATG TAG CC-3′ (SEQ ID NO: 39), 5′-GTT TTC AAA GTC ACTTCC G-3′ (SEQ ID NO: 40), 5′-GGT TAA CTC AAG CTG TGA AGC-3′ (SEQ ID NO:41), 5′-GGA GCT GAC ATG ACA TC-3′ (SEQ ID NO: 42), and 5′-GGC CAC GGTCAT GTT CAA GG-3′ (SEQ ID NO: 43).
 20. The method of claim 1, furthercomprising administering to the subject a pharmaceutically effectiveamount of a small molecule compound that reduces a COX-1 activity. 21.The method of claim 20, wherein the small molecule compound that reducesthe COX-1 activity is selected from the group consisting of Searle®SC-560,1-[(4,5-bis(4-methoxyphenyl)-2-thiazoyl)carbonyl]-4-methylpiperazinehydrochloride, valeryl salicylate, aspirin, dexketoprofen, keterolac,flurbiprofen, and suprofen.
 22. The method of claim 1, furthercomprising administering to the subject a pharmaceutically effectiveamount of a third compound that increases a COX-2 activity.
 23. Themethod of claim 22, wherein the third compound is selected from thegroup consisting of prostaglandin E; butaprost; sulprostone; Pfizer®CP-536,745-01; Pfizer® CP-043,305-02; Pfizer® CP-044,519-02; Pfizer®CP432; Ono Pharmaceutical® ONO-4819; Pfizer® CP-533,536; prostaglandinF_(2α); bimatoprost; cloprostenol; latanoprost; tafluprost; bonemorphogenetic protein-2; platelet derived growth factor; interleukin-1a;interleukin-1β; tumor necrosis factor-alpha; fibroblast growth factor,transforming growth factor-β; epidermal growth factor; parathyroidhormone; parathyroid hormone related peptide; and teriparatide.
 24. Themethod of claim 1, further comprising administering to the subject anultrasound therapy or exposing the subject to a pulsed electromagneticfield in an amount sufficient to increase a COX-2 activity in thesubject.